Immunoadhesin comprising a glycoprotein VI domain

ABSTRACT

The present invention provides a fusion protein comprising (1) the extracellular domain of glycoprotein VI or a variant thereof that is functional for binding to collagen and (b) the Fc domain of the immunoglobulin or a function-conservative part thereof, characterized by a polypeptide chain having an amino acid sequence as shown in FIG.  7  and whereby the fusion protein is obtainable by a process which provides the fusion protein in the form of a specific dimer.

PRIORITY CLAIM

This application is a continuation-in-part application of U.S.application Ser. No. 10/489,053, filed on Sep. 24, 2004, which is theNational Stage of International Application No. PCT/EP2003/005929, filedon Jun. 5, 2003, which, in turn, claims the benefit of EuropeanApplication No. 02 012 742.9, filed on Jun. 7, 2002, all of which areincorporated herein by reference.

The present invention relates to an immunoadhesin comprising a specificglycoprotein VI domain. The immunoadhesin of the invention is obtainableby a specific process providing the immunoadhesin in the form of adimer. The present invention also relates to the use of theimmunoadhesin of glycoprotein VI for the preparation of a medicament forthe prevention of intraarterial thrombosis in a specific group ofpatients. Moreover, the present invention relates to the use of theimmunoadhesin of glycoprotein VI for the preparation of a medicament forthe prevention and treatment of atheroprogression. The present inventionalso relates to the use of the immunoadhesin of glycoprotein VI for thepreparation of a medicament for the prevention and treatment of chronicprogression of atherosclerosis in diabetic patients. The presentinvention also relates to in vitro and in vivo screening methods for aninhibitor of GPVI mediated adhesion of platelets to active intravascularlesions.

Acute coronary or carotid syndromes are a major cause of death inWestern societies. Even in case of an initial survival of such acardiovascular event, many patents suffer from life-threateningcomplications such as intravascular thrombosis leading to furthermyocardial infarction or stroke.

Intravascular thrombosis is the result of aggregation of platelets in avessel whereby the blood flow in the vessel may be seriously reduced oreven completely inhibited. Specifically, the disruption of anatherosclerotic plaque initiates a cascade of events culminating inarterial thrombosis and ischemia of the downstream tissue, precipitatingdiseases such as myocardial infarction or ischemic stroke. The firstresponse to vascular injury is adhesion of circulating platelets toexposed subendothelial matrix proteins, which triggers subsequentplatelet aggregation. Among the macromolecular components of thesubendothelial layer fibrillar collagen is considered the mostthrombogenic constituent, as it acts as a strong activator of plateletsand supports platelet adhesion both in vitro and in vivo (1-3).

The platelet membrane proteins, which have been reported to be putativecollagen receptors, may be divided into those which interact indirectlywith collagen through collagen-bound von Willebrand factor (vWf),including GPIbα and the integrin α_(IIb)β₃, and those which interactdirectly with collagen including GPVI, the integrin α₂β₁, and CD38(reviewed in (2)). Only recently, the platelet glycoprotein VI (GPVI)has been identified as the major platelet collagen receptor (4). GPVI isa 60-65 kDa type I transmembrane glycoprotein, which belongs to theimmunoglobulin superfamily (5;6). In human and mouse platelets GPVIforms a complex with the FcR γ-chain at the cell surface (7;8). Ligandbinding to GPVI triggers tyrosine phosphorylation of the ITAM motif ofthe Fc receptor g chain initiating downstream signaling via Syk kinases,LAT, SLP-76, and phospholipase C (9-13). Platelets deficient in GPVIshow loss of collagen-induced adhesion and aggregation in vitro (4;14).Likewise, function blocking anti-GPVI monoclonal antibodies attenuate exvivo platelet aggregation in response to collagen and collagen-relatedpeptide CRP, which mimics collagen triple helix (15;16).

It is known that the problem of complications due to the aggregation ofplatelets can be addressed by administering inhibitors of plateletaggregation. For the treatment of acute coronary syndromes, GP IIb/IIIainhibitors such as ReoPro significantly improve the outcome of patients.However, a recent meta-analysis of clinical trials revealed asignificant remaining risk for death or myocardial infarction despiteoptimal antithrombotic intervention (Boersma E, Harrington R A,Moliterno D J, White H, Theroux P, Van de Werf F. de Torbal A, ArmstrongP W, Wallentin L C, Wilcox R G, Simes J, Califf R M, Topol E J, SimoonsM L. Platelet glycoprotein IIb/IIIa inhibitors in acute coronarysyndromes: a meta-analysis of all major randomised clinical trials.Lancet 2002; 359: 189-98). Specific severe side effects of thistherapeutic regimen are bleeding complications. These occurred in 2.4%of the patients with the most severe form of intracranial bleedingoccurring in almost 0.1% of the treated patents. Several mechanismshortcomings of the GP IIb/IIIa receptor blockade have been revealedwhich account for suboptimal effectively and side effects (Dickfeld T,Ruf A, Pogatsa-Murray G. Muller I, Engelmann B, Taubitz W, Fischer J,Meier O, Gawaz M. Differential antiplatelet effects of variousglycoprotein IIb-IIIa antagonists. Thromb Res. 2001; 101: 53-64. GawazM, Neumann F J, Schomig A. Evaluation of platelet membrane glycoproteinsin coronary artery disease: consequences for diagnosis and therapy.Circulation. 1999; 99: E1-E11).

The inhibition of platelet aggregation leads to a general impairment ofthe platelets with regard to their ability to aggregate. Accordingly,not only the undesired thrombosis formation is influenced, but also thegeneral ability of the platelets to terminate bleeding. Therefore, theadministration of inhibitors of platelet aggregation inherently leads tosevere side effects such as bleedings which may cause furtherlife-threatening complications. These side effects are of course stillmore problematic in patients suffering from diabetes.

Diabetes is one of the main risk factors for atherosclerosis.Additionally diabetes constitutes an increased risk of life threateningcomplications and excess morbidity in patients presenting with acutevascular and especially coronary syndromes. Diabetic patents withunstable angina present with a higher incidence of plaque ulceration andintracoronary thrombosis compared to non-diabetic patients.(Biondo-Zoccal G G L; Abbate A; Liuzzo G, Biasucci L: Atherothrombosis,inflammation, and diabetes. J Am Coll Cardiol 41; 1071-1077; 2003).

It is increasingly recognized that platelets are a major trigger for theprogression of atherosclerosis. The link between increasedatheroprogression, and increased platelet responsiveness and diabetes isso far an unresolved problem. Diabetic patients suffer from acutevascular complications independent of the degree of atherosclerosisindicative of different presently unknown mechanisms for plateletactivation in the development of diabetic acute vascular complicationsand atherosclerotic acute vascular complications.

Therefore, it is the problem of the invention to provide a medicamentwhich is useful for avoiding life-threatening complications subsequentto an acute coronary or carotid syndrome while maintaining the potencyof the blood for hemostasis.

It is a further problem of the present invention to provide a medicamentfor the treatment or prevention of atheroprogression.

It is a still further problem of the invention to provide a medicamentfor the treatment of diabetes, notably complications associated withdiabetes.

It is a further problem of the invention to provide an in vitro and anin vivo screening method for inhibitors of adhesion of platelets tointravascular lesions.

GENERAL DESCRIPTION OF THE INVENTION

The above problems are solved according to the claims. The presentinvention provides the first direct in vivo evidence indicating thatGPVI is in fact strictly required in the process of platelet recruitmentunder physiological shear stress following vascular injury. In differentmouse models of endothelial denudation both inhibition or absence ofGPVI virtually abolished platelet-vessel wall interactions and plateletaggregation, identifying GPVI as the major determinant of arterialthrombus formation. This indicates that inhibition of GPVI-ligandinteractions prevents arterial thrombosis in the setting ofatherosclerosis. The present invention uses the antithrombotic potentialof a specific soluble form of GPVI. Specifically, a fusion protein isprovided, which contains the extracellular domain of GPVI and a humanN-terminal Fc tag. The soluble form of human GPVI specifically binds tocollagen with high affinity and attenuated platelet adhesion toimmobilized collagen in vitro and to sites of vascular injury in vivo.Accordingly, the present invention is based on the recognition that theprecondition for intraarterial thrombosis as an acute clinicalcomplication is the initial adhesion of platelets to active lesions inthe vessel walls. The present inventors have recognised that plateletadhesion to subendothelial matrix collagen at a lesion of the vesselwall by the glycoprotein VI (GPVI) receptor represents the key event forthe formation of thrombosis. The inhibition of the adhesion of plateletsto subendothelial matrix collagen of the fusion protein of the inventionis therefore capable of not only preventing adhesion of platelets to anactive lesion, but also to prevent aggregation of platelets at theactive lesion. Thereby, the formation of intravascular thrombosis can beefficiently avoided without impairing the general ability of theplatelets for aggregation.

It is surprising that the complex process of the formation of thrombosismay be inhibited by the inhibition of a single platelet receptor in viewof the fact that different components of the subendothelial layers areligands and activators of platelets such as laminin, fibronectin, vonWillebrand factor (vWf) and collagen. Moreover, a wide variety ofreceptors on the platelets had been proposed by in vitro examinations,but the relevant receptor or receptor combinations which influenceadhesion of platelets to lesions in vivo had not been known before.

The present invention is also based on the recognition that GP VI is amajor meditor of platelet activity for the progression ofatherosclerosis. It is demonstrated that inhibition of thecollagen-medited GPVI activation attenuates atheroprogression inatherosclerosis prone Apo e −/− mice (see FIG. 16). Moreover, it isdemonstrated that the platelets from diabetic patients, who are alsoprone for advanced atherosclerosis and increased thromboticcomplications show an increased expression of the GPVI-coreceptorFc-receptor. Therefore platelets from diabetics might show increasedresponsiveness to collagen stimulation leading to the clinicallyobserved increased thrombotic complications in unstable angina, wherecollagen is uncovered from subendothelial vascular layers by plaquerupture or endothelial denudation.

The present invention provides therefore a treatment ofatheroprogression in patients, notably in patients suffering fromdiabetes. Moreover, the invention provides a medicament for thetreatment of acute vascular complications such as intravascularthrombosis especially in patients with diabetes. The immunoadhesinFc-GPVI-nt is a potent therapeutic tool to attenunate atheroprogressionand increased responsiveness of platelets to collagen via the GPVIreceptor. Therefore, Fc-GPVI-nt is a medicament for treatment ofatherosclerosis and particularly for the treatment of atheroscleroticcomplications in diabetes.

This invention provides a fusion protein (Fc-GPVI-nt) comprising thefollowing segments:

-   (a) the extracellular domain of glycoprotein VI (GP VI) or a variant    thereof that is functional for binding to collagen and-   (b) the Fc domain of an immunoglobulin or a function-conservative    part thereof.

The fusion protein is characterised by an amino acid sequence as shownin FIG. 7. The fusion protein according to the invention is obtained orobtainable by

-   (a) collecting 2 days after infection the culture supernatant of    Hela cells infected with an adenovirus for Fc-GPVI-nt coding for an    amino acid sequence as shown in FIG. 7;-   (b) centrifuging (3800 g, 30 min, 4° C.) the supernatant of step    (a);-   (c) filtrating (0.45 μm) the supernatant of step (b);-   (d) precipitating the immunoadhesin by addition of 1 vol. ammonium    sulfate (761 g/l) and stirring overnight at 4° C.;-   (e) pelletizing the proteins by centrifugation (3000 g, 30 min, 4°    C.),-   (f) dissolving the pelletized proteins of step (e) in 0.1 Vol PBS    and dialysed in PBS overnight at 4° C.;-   (g) clarifying the protein solution by centrifugation (3000 g, 30    min, 4° C.);-   (h) loading the solution of step (g) on a protein A column (HiTrap™    protein A HP, Amersham Pharmacia Biotech AB, Uppsala, Sweden);-   (i) washing the column with binding buffer (20 mM sodium phosphate    buffer pH 7.0, 0.02% NaN₃) until OD₂₈₀<0.01;-   (k) eluting fractions with elution buffer (100 mM glycine pH 2.7);-   (l) neutralizing the eluted fractions with neutralisation buffer (1    M Tris/HCl pH 9.0, 0.02% NaN₃);-   (m) pooling the fractions;-   (n) dialysing the pooled fractions in PBS overnight at 4° C.,-   (o) aliquoting the dialysed product and freezing at −20° C.

Under the above conditions, the fusion protein is obtained as acovalently linked dimer of a molecular mass of 160 kDa as measured undernon-reducing conditions by SDS-PAGE. Dimerisation of the fusion proteinpresumably occurs by inter-chain disulfide bonds of cysteins in aspecific domain adjacent to the GPVI fragment of the amino acid sequenceas shown in FIG. 7. The dimeric nature of the fusion protein depends atleast in part from the presence of a 1 g hinge region, and thepreparation process (non-reducing conditions). A monomeric fusionprotein is not useful as a therapeutic agent in practice since theinferior binding properties of a monomeric fusion protein as compared tothe dimeric fusion protein would require administration of protein in anamount which is in the order of one magnitude larger than the amount ofthe dimeric fusion protein for obtaining a similar effect, cf. FIG. 9(e). The administration of large amounts of protein is, however,problematic from a therapeutic and economic point of view, in particularin the treatment of chronic disease. For more information on theinvention, see below, including under the heading “Further Disclosure ofthe Invention”.

In embodiments the invention provides a dimeric fusion protein which isan immunoadhesin. It has a segment (a) that has the function of theextracellular domain of platelet GP VI. Said GPVI may be a mammalianGPVI, preferably it is human GPVI. Said function is preferably bindingto the GP VI ligand collagen. The whole extracellular domain of GPVI maybe used for said fusion protein or any fragments thereof provided saidfragments are capable of binding the collagen. A variant of the fusionprotein may have a modification at one or several amino acids of saidfusion protein (e.g. glycosylation, phosphorylation, acetylation,disulfide bond formation, biotinylation, chromogenic labelling likefluorescein labelling etc) Preferably, a variant is a homolog of saidfusion protein. An engineered variant may be tested easily for itscapability of binding to collagen using the methods disclosed herein.Most preferably, the polypeptide of residues 1 to 269 of FIG. 7 (SEQ IDNo. 1) is used as segment (a). However, said polypeptide may also bemodified by exchanging selected amino acids or by truncating saidsequence without abolishing said function.

Segment (b) of said fusion protein serves at least one of the followingpurposes: secretion of the fusion protein from cells that produce saidfusion protein, providing segment (a) in a form (e.g. folding oraggregation state) functional for binding collagen, affinitypurification of said fusion protein, recognition of the fusion proteinby an antibody, providing favourable properties to the fusion proteinwhen used as a medicament. Surprisingly and most importantly, segment(b) allows production of said fusion protein in mammalian, preferablyhuman, cells and secretion to the cell supernatant in active form, i ein a form functional for binding to collagen. Segment (b) may beantibody-derived, for example it may comprise an amino acid sequence ofan immunoglobulin heavy chain constant part, and is most preferably anFc domain of an immunoglobulin. Suitable immunoglobins are IgG, IgM,IgA, IgD, and IgE. IgG and IgA are preferred IgGs are most preferred,e.g. an IgG1. Said Fc domain may be a complete Fc domain or afunction-conservative variant thereof. A variant of Fc isfunction-conservative if it retains at least one of the functions ofsegment (b) listed above Most preferred is the polypeptide of residues273 to 504 of FIG. 7 (SEQ ID No. 1). It is, however, general knowledgethat such a polypeptide may be modified or truncated without abolishingits function.

Segments (a) and (b) of the fusion protein of the invention may belinked by a linker The linker may consist of about 1 to 100, preferably1 to 10 amino acid residues. The linker may be represented by residues270 to 272 of FIG. 7 (SEQ ID No 1), it may therefore comprise GlyGlyArg.

Most preferably, said fusion protein has the amino acid sequence of FIG.7 (SEQ ID No. 1) (termed Fc-GPVI-nt herein).

The invention further provides a nucleic acid sequence coding for thefusion protein of the invention. An embodiment of said nucleic acidsequence comprises a sequence selected from the following group:

-   (i) the nucleic acid sequence of FIG. 8 (SEQ ID No: 2) or a variant    thereof that codes for the same polypeptide according to the    degeneracy of the genetic code;-   (ii) a nucleic acid sequence coding for a polypeptide that has at    least 70% sequence homology to the polypeptide encoded by FIG. 8    (SEQ ID No: 2);-   (iii) a nucleic acid coding for a polypeptide of at least 300 amino    acids, whereby a segment of at least 100 amino acids is functional    for binding to collagen and a segment of at least 200 amino acids is    functional as an Fc domain; and-   (iv) a nucleic acid sequence coding for the fusion protein of claim    1.

The invention further provides a medicament for the prevention ortreatment of intraarterial thrombosis, containing a protein thatcomprises the extracellular domain of glycoprotein VI or a variantthereof that is functional for binding to collagen. Preferably, saidprotein is said fusion protein of the invention. If said medicamentcontains said fusion protein, said medicament preferentially furthercomprises a suitable carrier. Said medicament is preferably administeredparenterally, more preferably it is administered intravenously. As hasbeen found by the present inventors, GP VI-collagen interactions are themajor factor of platelet adhesion to an injured vessel wall. The fusionprotein of the invention can prevent binding of platelets toblood-exposed collagen in the vascular system by blocking saidblood-exposed collagen without inhibiting other platelet functions.

Alternatively, the medicament of the invention may contain a nucleicacid that codes for said fusion protein of the invention for genetherapy. Said nucleic acid preferably contains the nucleic acid sequencedefined above. Said nucleic acid is preferably contained in a vector,preferentially a viral vector. Vectors encoding said fusion protein maybe introduced into the vascular system of a patient such that e.g.endothelial cells are transduced therewith. Suitable vectors for genetherapy are known in the art. They may be based e.g. on adenoviruses, onadeno-associated viruses, on retro viruses, or on herpes simplexviruses. Vectors may be adopted for long-term or for short-termexpression of the fusion protein by transduced cells, as the patientrequires. The Fc domain of the fusion protein enables secretion of thefusion protein in active form by transduced cells.

The invention further provides a method of in vitro screening forinhibitors of binding of glycoprotein VI to collagen, comprising

-   (i) providing a surface that exposes collagen;-   (ii) contacting a portion of said surface with the fusion protein of    the invention under predetermined conditions that allow binding of    said fusion protein to said surface;-   (iii) contacting another portion of said surface with said fusion    protein in the presence of a test compound under conditions as in    step (ii);-   (iv) determining the amount of said fusion protein bound to said    surface in the absence and in the presence of said test compound;-   (v) identifying a test compound as inhibitor if binding of said    fusion protein to said surface is less in the presence of said test    compound as compared to the absence of the test compound; and-   (vi) optionally determining the functional effect of said inhibitor    on platelet aggregation and/or platelet activation.

The surface of step (i) may be a glass or plastic surface coated withcollagen. The portions of said surface may be the wells of a titer plateor a multi-well plate. A surface that exposes collagen may be easilyprepared by coating a glass or plastic surface with collagen asdescribed in the examples. Collagen-coated plates or multi-well platesare also commercially available. In step (ii), a predetermined amount ofsaid fusion protein is contacted with a first portion of said surfaceunder conditions (notably pH, buffer, temperature) that allow binding ofthe fusion protein to the surface. Preferably, conditions are chosenthat allow optimal binding to said surface. In step (iii), anothersurface portion is contacted with the same amount of fusion protein andunder the same conditions as in step (ii) in the presence of apredetermined amount or concentration of a test compound. More than oneamount or concentration of a test compound may be used. Said determiningof step (iv) preferably comprises washing of said surface portionscontacted according to steps (ii) and (iiii) one or more times in orderto remove unbound fusion protein. The amount of bound fusion protein maythen be determined e.g. by measuring the fluorescence of a fluorescentlabel (e.g. fluorescein, rhodamine etc.) attached to the fusion protein.Alternatively, bound fusion protein may be detected using an antibodyagainst said fusion protein, whereby said antibody may be fluorescentlylabelled. Alternatively, the antibody may be labelled with an enzyme(e.g. alkaline phosphatase, a peroxidase, luciferase) capable ofproducing a coloured or luminescent reaction product. Most conveniently,the fusion protein may be labelled with a chromogenic label such thatthe label changes its light absorption or light emission characteristicsupon binding to collagen. In this embodiment, washing off of unboundfusion protein is not needed.

In step (v), inhibitors may identified. Identified inhibitors orselected moieties thereof may be used as lead structures for improvementof the inhibitor. Such lead structures may be modified using chemicalmethods and the modified structures may again be tested with thisscreening method. Modified structures or test compounds with improvedinhibition properties may be selected and optionally further varied bychemical methods. In this way, iterative improvement of an inhibitor maybe achieved. The inhibitors identified using the screening methods ofthe invention are valuable as potential drugs against thrombosis andarteriosclerosis.

In step (vi), the functional effect of said inhibitor on plateletaggregation and/or platelet activation may be determined according tomethods described below, e.g. by intravital fluorescence microscopy.

Said screening method may be carried out on small, medium, or largescale depending on the number of test compounds to be tested. If manytest compounds are to be tested (e.g. libraries of chemical compounds),the screening method preferably takes the form of a high-throughputscreening (HTS). For HTS, the amount of bound fusion protein ispreferably detected using fluorescently labelled fusion protein.

The above screening method may also be adopted for screening forantibodies that inhibit binding of GP VI to collagen, notably antibodiesagainst the extracellular domain of GP VI. Such an antibody screeningmay be combined with e.g. hybridoma technology of generating monoclonalantibodies or any other antibody generating technique, whereby thefusion protein of the invention is preferably used as antigen.Antibodies in hybridoma cell supernatants may be used as said testcompounds.

The invention further provides antibodies produced by using the fusionprotein of the invention as immunogen. Moreover, use of an antibodyagainst GPVI is provided for the preparation of a medicament for theprevention of platelet adhesion at exposed subendothelial matrixcollagens in active atherosclerotic lesions as the initial trigger foracute coronary or carotid syndrome. Such indications may be diagnosed asdescribed below. Preferably, the patient is further characterized bysuffering from unstable atherosclerotic plaque. Said medicament ispreferably administered parenterally. Preferably, said antibodies aremonoclonal antibodies. Such antibodies may e.g. be prepared using thefusion protein of the invention as immunogen.

Furthermore, the invention provides a method of in vitro screening foran inhibitor of GPVI mediated adhesion of platelets to activeintravascular lesions, said method comprising the steps of

-   (i) providing a surface exposing collagen;-   (ii) contacting the surface with platelets under predetermined    conditions allowing for an adhesion of the platelets to the    collagen;-   (iii) measuring the adhesion of platelets in the presence of a test    compound; and-   (iv) identifying the test compound as an inhibitor of GPVI when the    adhesion of platelets to collagen is less in the presence of the    test compound as compared to the absence of the test compound; and-   (v) optionally determining the functional effect of said inhibitor    on platelet aggregation and/or platelet activation.

Platelets to be used in this method may be isolated according to knownprocedures (cf. example 7). They may be isolated from blood of mammalslike mice, rats, rabbits, pigs etc. Preferably, they are isolated fromhumans. Said platelets may be labelled e.g. with a fluorescent dye likefluorescein. The adhesion of platelets to said surface may be measuredas described in the examples. The test compounds for this method may besmall organic molecules. Preferably, the test compounds for this methodsare inhibitors identified in the above method of screening forinhibitors of binding of GP VI to collagen. In this way, the number ofcompounds to be screened using platelets can be significantly reducedand the likelihood of finding inhibitors functional with platelets canbe increased.

Method of in vivo screening for an inhibitor of GPVI mediated adhesionof platelets to active intravascular lesions, said method comprising thesteps of

-   (i) providing an in vivo model for active intravascular lesions;-   (ii) measuring the adhesion of platelets to an active intravascular    lesion in the presence of a test compound, and-   (iii) identifying the test compound as an inhibitor of GPVI when the    adhesion of platelets to the active intravascular lesion is less in    the presence of the test compound as compared to the absence of the    test compound.

Said in vivo model may be a suitable mammal like a mouse, a rat, arabbit etc. Preferably, it is a mouse. Platelets that are preferablyfluorescently labelled are introduced into the model prior to measuringthe adhesion of platelets to an active intravascular lesion in thepresence and in the absence of a test compound. Said test compound haspreferably been identified as an inhibitor in one of the above in vitroscreening methods. Adhesion of platelets to an active intravascularlesion may be carried out by using in vivo fluorescence microscopy asdescribed in example 8.

The present invention also provides a use of a fusion protein comprising

-   (a) the extracellular domain of glycoprotein VI or a variant thereof    that is functional for binding to collagen and-   (b) the Fc domain of an immunoglobulin or a function-conservative    part thereof, for the manufacture of a medicament for the treatment    of diabetes.

The fusion protein used for the manufacture of a medicament for thetreatment of diabetes is preferably a dimeric fusion protein in order toprovide for the possibility of dimerisation, a hinge region should bepresent in the fusion protein. The hinge region is required for allowingsuitable orientation of the polypeptide chains and formation ofinter-chain disulfide bonds Accordingly, the hinge region must have asufficient length and contain cysteine residues, preferably at least twocysteine residues. Preferably, the fusion protein comprises residues 1to 269 of SEQ ID No: 1. The fusion protein is used for the treatment ofacute complications of diabetes or for the treatment of chronicprogression of atherosclerosis in diabetic patients. Preferably, thefusion protein is Fc-GPVI-nt.

The treatment of diabetes using the fusion protein may include thetreatment of coronary or cardiovascular complications arising fromdiabetes for example atherosclerosis, thrombosis and other conditionsarising from collagen-GPVI interactions in diabetes sufferers.

The present invention also provides a method for the preparation of afusion protein of the invention (Fc-GPVI-nt), which comprises thefollowing steps:

-   (a) collecting 2 days after infection the culture supernatant of    Hela cells infected with an adenovirus for Fc-GPVI-nt coding for an    amino acid sequence as shown in FIG. 7;-   (b) centrifuging (3800 g, 30 min, 4° C.) the supernatant of step    (a);-   (c) filtrating (0.45 μm) the supernatant of step (b);-   (d) precipitating the immunoadhesin by addition of 1 vol. ammonium    sulphate (761 g/l) and stirring overnight at 4° C.;-   (e) pelletizing the proteins by centrifugation (3000 g, 30 min, 4°    C.),-   (f) dissolving the pelletized proteins of step (e) in 0.1 Vol PBS    and dialysed in PBS overnight at 4° C.;-   (g) clarifying the protein solution by centrifugation (3000 g, 30    min, 4° C.);-   (h) loading the solution of step (g) on a protein A column (HiTrap™    protein A HP, Amersham Pharmacia Biotech AB, Uppsala, Sweden);-   (i) washing the column with binding buffer (20 mM sodium phosphate    buffer pH 7 0, 0.02% NaN₃) until OD₂₈₀<0.01;-   (k) eluting fractions with elution buffer (100 mM glycine pH 2.7)-   (l) neutralizing the eluted fractions with neutralisation buffer (1    M Tris/HCl pH 9.0, 0.02% NaN₃);-   (m) pooling the fractions;-   (n) dialysing the pooled fractions in PBS overnight at 4° C.,-   (o) aliquoting the dialysed product and freezing at −20° C.-   The fusion protein of the present invention may also be expressed in    CHO cells.

DESCRIPTION OF THE FIGURES

FIG. 1 Platelet adhesion and aggregation following vascular injury ofthe common carotid artery in C57BL6/J mice in vivo. (a) Scanningelectron micrographs of carotid arteries prior to (left panels) and 2hrs after (right panels) vascular injury. Endothelial denudation inducesplatelet adhesion and aggregation, resulting in the formation of aplatelet-rich (lower left) thrombus. (b) Platelet-endothelial cellinteractions 5 min after vascular injury were investigated by in vivofluorescence microscopy of the common carotid artery in situ (blackcolumns). Animals without vascular injury served as controls (opencolumns). The left and right panels summarize transient and firmplatelet adhesion, respectively, of eight experiments per group.Platelets were classified according to their interaction with theendothelial cell lining as described²⁴ and are given per mm² of vesselsurface. Mean±s.e.m., asterisk indicates significant difference comparedto control, P<0.05. (c) Platelet aggregation following vascular injurywas determined by fluorescence microscopy in vivo (black columns).Animals without vascular injury served as controls (open columns).Mean±s.e.m., n=8 each group, asterisk indicates significant differencecompared to wild type mice, P<0.05. The microphotographs (right) showrepresentative in vivo fluorescence microscopy images in control animals(upper panel) or following vascular injury (lower panel). White arrowsindicate adherent platelets.

FIG. 2 inhibition of GPVI abrogates platelet adhesion and aggregationafter vascular injury. (a) Platelet adhesion following vascular injurywas determined by intravital videofluorescence microscopy. Fluorescentplatelets were preincubated with 50 μg/ml anti-GPVI (JAQ1) Fab fragmentsor control rat IgG. Platelets without mAb preincubation served ascontrol. The left and right panels summarize transient and firm plateletadhesion, respectively. Mean±s.e.m., n=8 each group, asterisk indicatessignificant difference compared to control, P<0.05. (b) illustrates thepercentage of platelets establishing irreversible adhesion after initialtethering/slow surface translocation is. (c) Platelet aggregationfollowing vascular injury in vivo. Aggregation of platelets preincubatedwith tyrodes, irrelevant rat IgG, or anti-GPVI Fab (JAQ1) was assessedby fluorescence microscopy as described. Mean±s.e.m., n=8 each group,asterisk indicates significant difference compared to control, P<0.05.(d) The photomicrographs show representative in vivo fluorescencemicroscopy images illustrating platelet adhesion in the absence orpresence of anti-GPVI Fab (JAQ1) or control IgG.

FIG. 3 Platelet adhesion following endothelial denudation inGPVI-deficient mice. (a) JAQ1-treated mice lack GPVI. Upper panels:Platelets from mice pretreated with irrelevant control IgG (left) oranti-GPVI (JAQ1) (right) were incubated with FITC-labeled JAQ1 andPE-labeled anti-mouse CD41 for 10 min at room temperature and directlyanalyzed on a FACScan™. A representative dot blot of 3 mice per group ispresented. Lower panel: Whole platelet lysates from three control IgG orJAQ1-treated mice were separated by SDS-PAGE under non-reducingconditions and immunoblotted with FITC-labeled JAQ1, followed byincubation with HRP-labeled rabbit-anti-FITC mAb. (b) Scanning electronmicrographs of carotid arteries 2 hrs after vascular injury in controlanimals (upper panels) or GPVI-depleted mice (lower panels). Endothelialdenudation induced platelet adhesion and platelet aggregation in controlanimals. In contrast, only very few platelets attached along the damagedvessel wall in GPVI-depleted mice. Subendothelial collagen fibers arevisible along the denuded area. (c) Platelet tethering and firm plateletadhesion, (d) transition from initial tethering to stable arrest(percentage of tethered platelets), and (e) platelet aggregationfollowing vascular injury of the carotid artery was determined inGPVI-deficient (JAQ1-pretreated mice) or control IgG-pretreated mice(for details see Materials and Methods). The panels summarize plateletadhesion (transient and firm) and platelet aggregation in eightexperiments per group. Mean±s.e.m., asterisk indicates significantdifference compared to control IgG, P<0.05. (f) The photomicrographsshow representative in vivo fluorescence microscopy images illustratingplatelet adhesion in GPVI-deficient (JAQ1) and control IgG-treated mice.

FIG. 4 Platelet adhesion to the surface of collagen coated glasscoverslips under physiological flow conditions was assessed ex vivo.Left panel: Platelets from mice pretreated with irrelevant control IgGimmunoadhesin (control) (left) or anti-GPVI immunoadhesin (Fc-GP VI-nt)(right) were investigated for adhesion under physiological flowconditions. The number of platelets was assessed by FACS counting of thewashed coverslips at the end of each experiment. Platelet tethering asthe first step of platelet adhesion was assessed after 30 seconds andfirm platelet adhesion after 5 min under flow conditions. (for detailssee Example 6). The panels summarize transient and firm plateletadhesion in eight experiments per group. Mean±s.e.m., asterisk indicatessignificant difference compared to control IgG, P<0.05.

FIG. 5 interaction of Fc-GP VI-nt with collagen was monitored in anELISA based assay. Adhesion of the immunoadhesin Fc-GP VI-nt consistingof the extracellular domain of GP VI and the FC part of an IgG tocollagen coated plates with increasing concentrations of Fc-GP VI-nt(0.5 μg to 10 μg) was investigated. The binding is visualized with asecondary antibody labelled with peroxidase directed to the Fc part ofFc-GP VI-nt Peroxidase is finally detected by ELISA. In thisrepresentative experiment binding of Fc-GP VI-nt to collagen wasmonitored with sufficient affinity, which reached saturation at μgconcentrations.

FIG. 6 interaction of the Fc-GP VI-nt with collagen and the possibilityto screen for GP VI inhibitors was demonstrated with the inhibitory antimouse GP VI antibody JAQ 1. Adhesion of the immunoadhesin Fc-GP VI-nt (2μg/well) to collagen coated ELISA plates is shown to be specific: theempty immunoadhesin Fc-nt did not show any binding. Thus, this providesan ELISA based assay for the screening against GP VI inhibitors with theupscale potential to high-throughput capacities.

FIG. 7 Amino acid sequence of Fc-GPVI-nt SEQ ID No: 1.

FIG. 8 DNA-Sequence of immunoadhesin Fc-GPVI-nt: SEQ ID No. 2. Bases 1to 807 encode the extracellular domain of GP VI. Bases 817 to 1515encode the Fc part of the IgG.

FIG. 9 Characterization of GPVI-Fc. (a) upper panel: Fc-GPVI-nt andcontrol Fc lacking the extracellular GPVI domain were used for SDS-PAGEunder reducing conditions. Coomassie blue stain (left) andimmunoblotting with peroxidase-conjugated goat anti-human Fc antibody(right) identified Fc-GPVI-nt with a molecular mass of −80 kDa. Middlepanel: immunoblotting of Fc, Fc-GPVI-nt, or human platelets using theanti-GPVI monoclonal antibody 5C4. 5C4 detected both adenovirallyexpressed Fc-GPVI-nt fusion protein and platelet GPVI, but not thecontrol Fc. Lower panel. Molecular mass under reducing (right) andnon-reducing (left) conditions. While the molecular mass of Fc-GPVI-ntwas approximately 80 kDa under reducing conditions, the complete nt with−160 kDa protein was identified under non-reducing conditions. (b-d)Characterization of Fc-GPVI-nt collagen interactions. (b) Binding assaysusing different concentrations of soluble Fc-GPVI-nt and immobilizedcollagen (10 μg/ml) were performed to define Fc-GPVI-nt-collageninteractions. Bound Fc-GPVI-nt was detected by anti-Fc mAb antibody(dilution 1:10.000) and is given relative to the binding observed at 10μg/ml Fc-GPVI-nt Fc-GPVI-nt binds to collagen in a saturable manner.Mean±s.e.m., n=6 each Fc-GPVI-nt concentration, asterisk indicatessignificant difference compared to 0 μg/ml Fc-GPVI-nt, P<0.05. (c, leftpanel shows binding of Fc-GPVI-nt (20 μg/ml) to various substrates.Binding of Fc-GPVI-nt to BSA (10 μg/ml) or vWF (10 μg/ml) is given aspercentage of GPVI-dimer-binding to immobilized collagen. Binding ofFc-GPVI-nt did not occur to BSA or vWF, supporting the specificity ofFc-GPVI-nt binding. Mean±s.e.m., asterisk indicates significantdifference compared to collagen, P<0.05. (c, right panel) illustratesbinding of Fc-GPVI-nt (20 μg/ml) or Fc (20 μg/ml) to immobilizedcollagen (10 μg/ml). Bound Fc-GPVI-nt or Fc was detected by anti-Fc mAbantibody (dilution 1:10.000) and is given relative to the bindingobserved with Fc-GPVI-nt. Only Fc-GPVI-nt, but not Fc or anti-Fc mAbbinds to immobilized collagen. Mean±s.e.m., n=8 each group, asteriskindicates significant difference compared to Fc-GPVI-nt binding, P<0.05.(d) Fc-GPVI-nt (20 μg/ml) was preincubated for 10 min with differentconcentrations of soluble collagen. After incubation the plates werewashed and Fc-GPVI-nt binding was detected by peroxidase-conjugated goatanti-human IgG antibody (dilution 1:10.000). Fc-GPVI-nt binding is givenrelative to the binding observed in the absence of soluble collagen.Soluble collagen inhibits GPVI-Fc-dimer-dimer binding to immobilizedcollagen in a dose-dependent manner. Mean±s.e.m., n=3 each collagenconcentration, asterisk indicates significant difference compared to 0mg/ml collagen, P<0.05. (e) The difference of the binding affinitybetween the monomeric form of the GPVI-Fc fusion portein and Fc-GPVI-ntwas assessed in direct comparison. The binding of the monomer and dimerwas assessed on collagen type 1 coated ELISA plates. Increasingconcentrations of the GPVI fusion proteins bond to collagen in asturable manner. Here a Linewaver Burke plot is demonstrated foraffinity assessment (e). The affinity of the monomeric GPVI fusionprotein was about 10 times lower compared to equimolar concentrations ofthe dimeric form Fc-GPVI-nt

FIG. 10 Fc-GPVI-nt inhibits CD 62 P activation on human platelets as aparameter of release of intracellular transmitter substances from alphagranules by increasing doses of collagen. Human platelets were isolatedfrom whole blood and incubated with anti-CD 62 antibodies labelled withPE (for details see Material and Methods). Fluorescence was determinedin a Becton Dickenson FACS device. Representative histogramms are shown.Increasing concentrations of collagen from 0 to 10 μg/ml induced a shiftof fluorescence in the presence of the control Fc protein (100 μg/ml;blue line). In the presence of Fc-GPVI-nt (100 μg/ml; red line), theshift of fluorescence and hence CD 62 P activation was markedlyinhibited.

FIG. 11 Specific inhibition of collagen-mediated platelet aggregationand release of endogenous transmitters from dense and alpha granules byFc-GPVI-nt. (a) Human platelets were incubated with control Fc (80μg/ml) or Fc-GPVI-nt (80 μg/ml). Aggregation of platelets was inducedwith collagen (1 μg/ml) or ADP (5 μM) or TRAP (10 μM) and aggregationwas determined in an aggregometer under stirring conditions (for detailssee Material and Methods). Triplet measurements from n=5 different blooddonors were carried out. The means±s.e.m are given in % aggregation ofthe control aggregation without fusion proteins. (b) ATP release wasmeasured simultaneously in the same probes after incubation with controlFc (80 μg/ml) or Fc-GPVI-nt (80 μg/ml). The amount of ATP release isgiven in % of controls without fusion protein. (c) PDGF release wasdetermined in human platelets with an ELISA system specific for humanPDGF under basal conditions and after collagen (20 μg/ml) stimulation(for details see Material and Methods). Preincubation with control Fchad no significant effect on PDGF release from collagen-stimulatedplatelets, whereas Fc-GPVI-nt (100 μg/ml) reduced the PDGF releasesignificantly. Inhibition of PDGF release did not occur in unstimulatedplatelets.

FIG. 12 Fc-GPVI-nt has no significant effect on bleeding time in humanblood ex vivo. Bleeding time in human blood was measured ex vivo afterADP/collagen stimulation and epinephrine/collagen stimulation in aPFA-100 device. Fc-GPVI-nt (5 and 20 μg/ml) and Fc (5 and 20 μg/ml) didnot prolong bleeding time whereas ReoPro^(R) in a therapeuticallyrelevant concentration (5 μg/ml) maximally prolonged bleeding time underboth conditions. The means±s.e.m. from n=4 blood donors with tripletmeasurements are summarized.

FIG. 13 Fc-GPVI-nt inhibits platelet adhesion to immobilized collagenunder flow conditions. Human platelets (2×10⁸ cells/ml) were isolatedfrom whole blood (for details see “materials and methods”). Plates werecoated with immobilized collagen (10 μg/ml) or vWF (10 μg/ml). Plateletadhesion to the coated plates was determined in a parallel plate flowchamber in the presence of Fc-GPVI-nt or Fc lacking the extracellularGPVI domain (200 μg/ml). Inhibition of platelet adhesion by Fc-GPVI-ntis given in % of control (Fc control). Fc-GPVI-nt significantlyattenuated platelet adhesion on immobilized collagen at shear rates of500 sec⁻¹, and 1000 sec⁻¹, respectively. In contrast, Fc-GPVI-nt did notaffect platelet adhesion on immobilized vWF. Mean±s.e.m., n=4 eachgroup, asterisk indicates significant difference compared to control Fc,P<0.05. The lower panels show representative microscopic images.

FIG. 14 Fc-GPVI-nt has favourable pharmacokinetics with a prolongedplasma half life after intraperitoneal injection in mice in vivo. Bloodconcentrations of Fc-GPVI-nt were determined with specific anti-Fcantibodies and ELISA (for details please see “material and methods”).(a) Single intraperitoneal injection of Fc-GPVI-nt (4 μg/g) led to rapidpeak blood concentrations of Fc-GPVI-nt after ˜24 h with slow decline ofFc-GPVI-nt blood concentrations. The means±s.e.m. from 10 animals aredemonstrated. (b) Repeated intraperitoneal applications (10 μg/g; twiceweekly) leads to continuous accumulation of Fc-GPVI-nt in mice in vivoover 28 days. The means±s.e.m. from 6 animals are demonstrated. (c)intravenous single dose injection of 30 μg Fc-GPVI-nt (1 μg/g bodyweight); 60 μg (2 μg/g body weight) and 100 μg Fc-GPVI-nt (3 μg/g bodyweight) per mouse led to a dose-dependent increase of immunoadhesinplasma concentration. The plasma concentration in the two higher dosesin these mice in vivo reached prolonged elevated levels from 5 to 60minutes and after 24 hours, sufficient for effective collagen scavengingand therefore effective inhibition of GPVI receptor activation onplatelets. The means±s.e.m. from 5 animals are demonstrated.

FIG. 15 Effects of Fc-GPVI-nt on platelet adhesion and aggregation invivo. (a) Mice (n=6 per group) were treated with 2 mg/kg or 4 mg/kgFc-GPVI-nt iv. Integrilin (0.2 mg/kg)-treated mice served as positivecontrols (n=8). Bleeding times were determined as described (see“materials and methods”). The Fc-GPVI-nt fusion protein did not increasetail bleeding times compared to control animals. In integrilin-treatedmice tall bleeding time was massively prolonged. **P<0.05 vs. control.(b) inhibition of GPVI abrogates platelet adhesion and aggregation aftervascular injury. Platelet adhesion following vascular injury wasdetermined by intravital video fluorescence microscopy. Mice werepretreated with 1 or 2 mg/kg Fc-GPVI-nt or equimolar amounts of controlFc. The left and right panels summarize platelet tethering and firmplatelet adhesion, respectively. Mean±s.e.m., n=5 each group, asteriskindicates significant difference compared to Fc, P<0.05. (c) Effects ofFc-GPVI-nt on thrombus formation following vascular injury in vivo. Thenumber of platelet thrombi (right) and the total thrombus area (left)were assessed by fluorescence microscopy as described. Mean±s.e.m., n=5each group, asterisk indicates significant difference compared to Fc,P<0.05. (d) The photomicrographs show representative in vivofluorescence microscopy images illustrating platelet adhesion in theabsence or presence of 1 or 2 mg/kg Fc-GPVI-nt or control Fc. Barsrepresent 50 μm. (e) Scanning electron micrographs of carotid arteries 1hr after vascular injury in Fc- or Fc-GPVI-nt treated animals.Endothelial denudation induced platelet adhesion and plateletaggregation in Fc-treated mice. In contrast, only very few plateletsattached along the damaged vessel wall in Fc-GPVI-nt-treated mice.Subendothelial collagen fibers are visible along the denuded area. Barsrepresent 10 μm (f) Fc-GPVI-nt specifically binds to the subendotheliumof carotid arteries. The binding of Fc-GPVI-nt to the subendothelium wasdetermined on carotid sections, stained with peroxidase-conjugated goatanti-human IgG antibody. Carotid arteries obtained from Fc-treated miceserved as controls. Fc-GPVI-nt but not Fc control protein was detectedat the subendothelium, as indicated by the brown staining. Originalmagnification: 100-fold.

FIG. 16 Fc-GPVI-nt significantly attenuates atheroprogression apo e −/−knockout mice in vivo. Apo e −/− mice were treated with Fc-GPVI-nt (4μg/g) or control Fc (4 μg/g) intraperitoneally for 4 weeks twice weekly.Atheroprogression was investigated post mortem after sudan red stainingof the large vessels to visualise atheroma and plaque formation. Incontrol animals extensive plaque formation of carotid arterypreparations was indicated by the red colour in particular in thebranching region. In Fc-GPVI-nt treated animals atherosclerosis wasalmost completely abolished in carotid arteries of apo e −/− mice.Representative macroscopic whole vascular preparations of the carotidearteries of an apo e −/− mouse after 4 weeks treatment with Fc-GPVI-nt(left side) and of an apo e −/− mouse after 4 weeks treatment with thecontrol Fc protein (right side) are shown.

FIG. 17 Freshly isolated platelets from patients suffering from diabetesmellitus show reduced expression of the fibrinogen receptor (CD61, top)and increased expression of the Fc receptor (CD32, middle) and thereforeincreased expression of GPVI. The correlation between CD32 expressionand GPVI expression (detected by the specific monoclonal antibody 4C9)is shown on human platelets (bottom). Human platelets were isolated fromwhole blood from patients suffering from diabetes and incubated withfluorescent anti-CD61 and anti CD32 antibodies or FITC labelled 4C9antibodies. Fluorescence was determined in a Becton DickensonFACScalibur device. The means+/−s.e.m. from n=111 diabetic patients andfrom n=363 patients without diabetes are summarized. Correlation of CD32fluorescence and 4C9 fluorescence was calculated with the correlationcoefficient r=0.516.

FIG. 18 Amino acid sequence of a monomeric fusion protein based onFc-GPVI-nt

FIG. 19: The fusion protein of an embodiment of the present invention iscompared with alternative thrombus inhibitory molecules. It is shown theGPVI-Fcnt reduces the platelet thrombus formation in vivo in comparisonwith an Fc molecule (control) and a GPVI-Fc fusion protein which doesnot comprise a linker between the extracellular domain of GPVI and theFc portion.

FIG. 20: Plaque size in the aortae of 12-14 week old cholesterol-fedApoE-1-mice which have been treated with various proposedanti-thrombotic agents, including the fusion protein of an embodiment ofthe present invention.

FIG. 21: Adhesion of platelets in response to endothelial injury invivo.

FIG. 22: Adhesion of platelets to atherosclerotic plaques in vivo.

FIGS. 23 to 25: Fc-GPVI-nt and Fc (a control protein) were generated andpurified as described. A competing Fc-GPVI-nt with another specificlinker according to the sequence alluded to in WO 01/00810 was clonedaccording to the same method. For this purpose, a similar construct wasgenerated using the same DNA sequence as described in FIG. 7 except forthe Gly-Gly-Arg linker on amino acid positions 270-272, which wasreplaced by a Ala-Ala-Ala linker, amplified and purified according tothe same methods.

Thrombus Formation In Vivo

Preparation and Measurement of Thrombus Formation in the Carotid Artery

Vascular injury was induced in the mouse common carotid artery. C57BL6/Jmice were anesthetized by intraperitoneal injection of a solution ofxylazine (5 mg/kg body weight, AnaSed; Lloyd Laboratories) and ketamine(80 mg/kg body weight, Ketaset; Aveco Co, Inc). A catheter wasintroduced into the tail vein, and either Fc-GPVI-nt or control Fc wereinjected at doses of 2 mg/kg body weight, and flushed with 200 μLsaline. Then, the right cervical region was dissected free between thesternohyoid and myohyoid muscle, a clamping and subsequent ligation ofthe cranial internal carotid artery was carried out. Then, the commoncarotid and the external carotid were clamped. The right internalcarotid artery was incised. A thin coronary guiding wire of defined sizewas introduced into the right common carotid artery and a vascularinjury was induced by moving the wire up and down three times. This ledto complete loss of endothelial cells at the site of injury.

Thereafter, the animas were allowed to sit up and survived for 4 hoursafter the intervention. Then, anaesthesia was reinduced, and the animalswere perfused with 10 mL of saline solution through their tail veins,followed by a second infusion of 4 mL paraformaline (PFA, 4%). Then, thelarge vessels including both carotid arteries (including the bifurcationand the common carotids up to 3.5 mm proximal to the carotidbifurcation) were dissected free and taken out. Fat tissue was removed.The vessels were then opened longitudinally and spread out onmicroscopic cover slips.

The en face thrombus size was measured by digitalized imaging analysis,which measured the relative thrombus area as % the total vessel area asspread out on the slide.

Statistics

All groups were compared by analysis of variance (ANOVA) using aBonferroni's post-hoc analysis.

2. Results:

1. Aggregation and ATP Release

Aggregation and ATP release measurements of freshly isolated humanplatelets in response to collagen (1 μg/mL) resulted in strongphotometric signals, as described The results of aggregation are shownin FIG. 23 as % of the internal standard of the aggregometric device.This aggregation was slightly but not significantly less in the presenceof the control Fc, and slightly more reduced in the presence of theFc-GPVI-nt containing the Ala-Ala-Ala linker. In contrast,administration of the Fc-GPVI-nt containing the Gly-Gly-Arg linker atthe same concentration (40 μg/mL) markedly and highly significantlysuppressed aggregation. FIG. 23 shows results from four independentexperiments. *p<0.05 vs. controls.

FIG. 24 shows the results for ATP release, which correspond well to thefindings for aggregation.

FIG. 24: ATP Release

Mean ATP release form human platelets in response to 1 μg/mL collagen(expressed as % of the internal standard) in the presence of eitherconstruct. ATP release was slightly but not significantly reduced in thepresence of the control Fc, and slightly more reduced in the presence ofthe Fc-GPVI-nt containing the Ala-Ala-Ala linker. In contrast,administration of the Fc-GPVI-nt containing the Gly-Gly-Arg linker atthe same concentration (40 μg/mL) markedly and highly significantlysuppressed ATP release

Shown are results from four independent experiments. *p<0.05 vs.controls.

FIG. 25: Endothelial Lesion In Vivo and Determination of en FaceThrombus Size.

Mice were injected with 2 mg/kg of either construct, then endotheliallesions were induced. The right carotid arteries were excised, andprepared as described The en face thrombus size was related to theoverall surface of the spread-out vessel and expressed as %. FIG. 25shows the results from five independent experiments in five independentanimals. It is obvious that administration of the Fc-GPVI-nt (Procorde)construct containing the specific Gly-Gly-Arg linker markedly andsignificantly inhibits thrombus formation at the endothelial lesion ofthe right carotid artery, where the lesion had been placed. In contrast,the administration of the Fc-GPVI-nt construct containing the specificAla-Ala-Ala linker resulted in only a trend towards decreased thrombusformation, which did not reach statistical significance.

The uninjured left carotid arteries were investigated as controls in allanimals and did not show any platelet adhesion or thrombus formation.*p<0.05 vs. Fc controls.

The uninjured left carotid arteries were investigated as controls.

DETAILED DESCRIPTION OF THE INVENTION

A previous hypothesis suggested that platelet glycoprotein (GP) lbbinding to von vWf recruits flowing platelets to the injured vessel wall(Ruggeri, Z. M.: Mechanisms initiating platelet thrombus formation.Thromb. Haemost 1997; 78, 611-616), whereas subendothelial fibrillarcollagens support firm adhesion and activation of platelets (van Zanten,G. H. et al. Increased platelet deposition on atherosclerotic coronaryarteries. J Clin. Invest 1994; 93, 615-32; Clemetson, K. J. & Clemetson,J. M. Platelet collagen receptors. Thromb. Haemost 2001, 86, 189-197).However, the present invention demonstrates by in vivo fluorescencemicroscopy of the mouse carotid artery that inhibition or absence of themajor platelet collagen receptor, GPVI, instead, abolishesplatelet-vessel wall interactions following an endothelial erosion.Unexpectedly, inhibition of GPVI reduces platelet tethering and adhesionto the subendothelium by approximately 89%. Furthermore, stable arrestand aggregation of platelets is virtually abolished under theseconditions. The strict requirement for GPVI in these processes wasconfirmed in GPVI-deficient mice, where platelets also fall to adhereand aggregate on the damaged vessel wall. These findings reveal anunexpected role of GPVI in the initiation of platelet attachment atsites of vascular injury and unequivocally identify platelet-collageninteractions as the major determinant of arterial platelet-inducedatherosclerotic complications.

The fact that GP VI generally functions as a receptor for thesubendothelial matrix collagen has been described (Morol M, Jung S M,Okuma M, Shinmyozu K. A patient with platelets deficient in glycoproteinVI that lack both collagen-induced aggregation and adhesion. J Clininvest 1989; 84: 1440-1445). These authors characterized platelets invitro originating from patients with a GP VI receptor deficiency.However, the physiological significance of the interaction of collagenand GP VI receptor in the in vivo context and the relative contributionof the GP VI receptor for adhesion following vascular injury wasunknown. In particular, it was not known that inhibition of thisreceptor inhibits the key step in the formation of intravascularthrombosis that is platelet tethering. The present invention reveals theGP VI receptor as an essential receptor for platelet adhesion to thesubendothelium via the attachment to subendothelial matrix collagen invivo. Amongst the variety of other platelet surface proteins such as GPIb (von Willebrand receptor), the αIIbβ3 integrin receptor, the α2β1integrin or the GP V receptors, we have surprisingly identified the GPVI receptor to be an essential receptor to mediate platelet adhesion tothe vascular wall. Since platelet adhesion is the first and mostimportant step for platelet aggregation and intraarterial thrombusformation under physiologic shear stress conditions, the followingdeleterious effects leading to intraarterial occlusion are thefunctional basis for the clinical syndromes of myocardial infarction orcerebral stroke. In a chronic setting, the interaction of platelets withthe endothelium propagates early steps of arteriosclerosis. Ourinvention also showed for the first time that the GP VI receptor plays acrucial role amongst the complex variety of several platelet surfaceproteins for initial platelet adhesion and for chronicalplatelet-endothelium interaction in the propagation of arteriosclerosis.

WO 01/16321 and WO 01/00810 disclose a DNA and protein sequence of thehuman GPVI receptor. However, the significance on platelet adhesion andactivation by endothelial lesions has not been demonstrated in an invivo background.

U.S. Pat. No. 6,383,779 discloses fusion proteins of GPVI. However, thisreference does not disclose a dimeric fusion protein or any therapeuticeffect of GPVI.

Recently, the different phases of platelet-collagen interaction toartificial collagen in vitro during perfusion conditions wereinvestigated (Moroi M, Jung S M, Shinmyozu K, Tzomlyama Y, Ordinas A andDiaz-Ricart M. Analysis of platelet adhesion to collagen-coated surfaceunder flow conditions: the involvement of glycoprotein VI in theplatelet adhesion. Blood 1997; 88: 2081-2092). The authors of that studyalready pointed out the importance of collagen-GP VI interaction duringshear stress conditions. However, the relevance of subendothelial matrixcollagen for the adhesion could not be studied in this artificial invito situation. As a consequence of limited relevance of their in vitromodel, the authors of the above mentioned study came to the conclusionthat GP VI receptors are rather involved in platelet activation than inplatelet adhesion to the endothelium. In contrast, the von Willebrand GPIb receptor is significantly involved in platelet-subendothelialinteraction. These authors also focussed all available information aboutplatelet-collagen interaction in a review of the current literature.Previously, Moroi M and Jung, S M (Platelet receptors for collagen.Thromb. Haemost 1997; 78: 439-444) have discussed collagen fibrilinteraction with different collagen receptors on platelets for theadhesion and thrombus formation of platelets. However, the authors didnot expect a relevant role of the GP VI receptor for the adhesion in aclinically relevant in vivo situation as they could not validate thesignificance of the different collagen receptors to the adhesionprocess.

Therefore, the present invention provides a solution to the problem ofinhibiting the relevant target for the platelet-subendothelialinteraction and for platelet adhesion without provoking undesired sideeffects of bleeding complications. Besides the well known interaction ofcollagen-platelet via the GP VI receptor, we could provide data for theinteraction of the native subendothelial matrix and platelets measuredby in vivo platelet adhesion. Consecutively, we could validate thesignificance of the GP VI-endothelium interaction for platelet adhesionas initial step of intravascular thrombosis. Thus, our invention solvesthe problem of an effective antiplatelet drug treatment for theimportant step of platelet adhesion without undesired side effects.

Further, the invention provides an immunoadhesin (the fusion protein ofthe invention). In a specific embodiment, the immunoadhesin consists ofthe extracellular domain of the GP VI receptor together with the Fc partof an IgG immunoglobulin (Fc-GPVI-nt). This novel fusion protein isbased approximately 50% on the original DNA sequence of GP VI aspublished previously. The protein structure of the immunoadhesin isnovel as the recombinant fusion protein does not form a membrane proteinlike the GP VI receptor but is a soluble, immunoglobulin-likeimmunoadhesin released by the respective host cell. This immunoadhesincan block the ligand-receptor interaction of collagen and GP VI. Ourresults demonstrate that the immunoadhesin has marked effects on themain physiological functions of platelets induced by collagenstimulation. Collagen-induced aggregation, adhesion and the releasefunction can be inhibited by the immunoadhesin to the same extent asdoes a specific, monoclonal antibody. The mechanism, however, isdifferent whereas the antibody inhibits GP VI activation by directlybinding to the ligand binding site of the GP VI receptor, theimmunoadhesin scavenges the GP VI ligand collagen and therefore preventsligand-mediated GP VI activation.

The immunoadhesin of the invention is a novel GP VI inhibitor. It hasthe advantage of selective inhibition of the activated branch of GP VImediated effects by ligand scavenging. Secondary effects, like antibodymediated effects on GP VI receptor internalisation are prevented.Fc-GPVI-nt can be used for the treatment of atheroscleroticcomplications caused by unstable atheroslerotic plaques with plaquerupture or endothelial lesion. Therefore, the immunoadhesin Fc-GPVI-ntserves as a therapeutic inhibitor for collagen-mediated GP VI activationwithout affecting the intrinsic activity of the GP VI receptor with therelevant signalling system.

Moreover, the GP VI immunoadhesin serves as an ideal epitope forantibody selection. The Fc part allows the convenient purification ofthe protein and simple fixation to surfaces to perform large scaleantibody selection against antibody libraries i.e. by phage display. Theselection allows selective antibody screening to the relevant epitopethat resembles the intact protein with a similar structure as the nativeprotein.

Finally, the Fc-GPVI-nt is an important tool for the screening forinhibitors of GP VI receptor activation. We have established anELISA-based in vitro assay simulating the collagen GP VI interaction bycollagen precoated plates as the ligand. This assay can alternatively berun with fluorescence-labelled Fc-GPVI-nt and thus be upscaled tohigh-throughput formats. This assay allows for the screening of both,inhibitory antibodies or small molecules for their potency to inhibit GPVI function by fluorescence measurement With this cell free screeningassay, a prototype method for a high-throughput-scaleable fluorescencescreening assays for drug testing has been established.

Based on the recent improvements in imaging techniques by intravascularultrasound or nuclear magnetic resonance imaging, it is possible toidentify patients with atherosclerosis being at risk of acute clinicalcomplications such as acute coronary or carotid syndrome, whereby thepatents have active lesions as possible causes for intravascularthrombosis. It is then possible by the present invention to prevent theformation of intravascular thrombosis by the administration of amedicament containing an antibody against platelet glycoprotein VI(GPVI) without undesired side effects.

Active lesions are characterized by the unmasking of subendothelialmatrix collagens and platelet activation. The occurrence of such lesionscan be investigated e.g. by intravascular ultrasound or thermography(e.g., Fayed and Fuster, Clinical imaging of the high-risk or vulnerableatherosclerotic plaque. Circulation 2001; 89:305-316) or nuclearresonance imaging (Helft et al., Progression and Regression ofAtherosclerotic Lesions. Circulation 2002; 105: 993-998). Such lesionsare highly probable in patients with acute coronary or carotidsyndromes, and the risk of the reoccurrence of acute clinicalcomplications such as myocardial infarction or stroke is very high,decreasing progressively with increasing time distance from the primaryevent.

Therefore, the present invention also provides a method of treating apatient suffering from an acute coronary or carotid syndrome, saidmethod comprising for avoiding intravascular thrombosis the steps of

-   (a) determining the presence or absence of active intravascular    lesions in the patient; and-   (b) treating the patient with an antibody against platelet    glycoprotein VI (GPVI) in case of the presence of intravascular    lesions.

Moreover, based on the present invention, it is possible to treatpatients being at risk of intravascular thrombosis due to the rupture ofcomplex arteriosclerotic plaques. The rupture also unmasks thesubendothelial collagen matrix. As a consequence of intraarterialthrombus formation, the perfusion of vital organs is blocked with theabove described important and life threatenting clinical syndromes.

The present invention also provides a method of treating a patientsuffering from a chronic atherosclerotic syndrome, said methodcomprising for avoiding intravascular thrombosis the steps of

-   (a) determining the presence or absence of the onset of    atheroprogression in the patient; and-   (b) treating the patient with an antibody against platelet    glycoprotein VI (GPVI) in case of the presence of intravascular    lesions.

Accordingly, based on the present invention, it is possible to treatpatients being at risk of atherosclerosis. In order to preventatheroprogression, a patient is treated with the fusion protein of theinvention in order to prevent interaction between platelets and exposedsubendothelial collagen. The fusion protein of the invention blocks theligand for the GPVI platelet receptor in the vascular wall (e.g.subendothelium) so that an interaction between the platelets and exposedcollagen is inhibited.

The fusion protein of the invention may be in the form of a lyophilisedpowder which is dispersed in a suitable pharmaceutically acceptableliquid carrier prior to administration to a patient. The fusion proteinof the invention can also be incorporated into pharmaceuticalcompositions suitable for parenteral, in case of the treatment of acutecomplications preferably intraarterial or intravenous administration.Such compositions usually comprise the fusion protein and apharmaceutically acceptable carrier. A pharmaceutically acceptablecarrier includes solvents, dispersion media, antibacterial andantifungal agents and isotonic agents, which are compatible withpharmaceutical administration. The present invention includes methodsfor manufacturing pharmaceutical compositions for the treatment ofchronic or acute cardiovascular disease. Such methods compriseformulating a pharmaceutically acceptable carrier with the fusionprotein of the invention. In case of the treatment of acutecardiovascular disease, the composition is preferably administeredintravenously or intraarterially. In case of the treatment of chroniccardiovascular disease, the composition may also be administeredsubcutaneously and intraperitoneally. Such compositions can furtherinclude additional active compounds, such as further polypeptides (suchas insulin) or plktherapeutically active small molecules. Thus, theinvention further includes methods for preparing a pharmaceuticalcomposition by formulating a pharmaceutically acceptable carrier withthe fusion protein of the invention and one or more additional activecompounds such as insulin. In case of the coformulation of the fusionprotein and insulin for the treatment of diabetic patients, it ispreferred that the dosage form allows separate storage of the differentproteins whereby mixing of the proteins is carried out just prior orduring the administration of the composition. Accordingly, applicationby a multi-chamber syringe is considered. A pharmaceutical compositionof the invention is formulated to be compatible with its intendedparenteral route of administration. Examples of routes of parenteraladministration include, e.g., intraarterial and intravenousadministration. Solutions or suspensions used for parenteral may includea sterile diluent such as water for injection, saline solution,polyethylene glycols, fixed oils, glycerine, propylene glycol, TWEEN orother synthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; chelating agents such as ethylenediaminetetraaceticacid; antioxidants such as ascorbic acid or sodium bisulfite; bufferssuch as acetates, citrates or phosphates and agents for the adjustmentof tonicity such as sodium chloride, dextrose, saccarose or mannitose.The pH can be adjusted with acids or bases, such as hydrochloric acid orsodium hydroxide. The parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic. Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, or phosphate buffered saline(PBS). The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol), and suitable mixtures thereof.The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of micoorganisms can be achieved by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin. Sterile injectable solutionscan be prepared by incorporating the active compound (e.g., apolypeptide or antibody) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich containssss a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. It is especiallyadvantageous to formulate oral or parenteral compostions in dosage unitform. A dosage unit form are discrete units suited as unitary dosagesfor a patient. Each unit contains a predetermined quantity of activecompound to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. A therapeutically effective amountof fusion protein (i.e., an effective dosage) for the treatment of acutecomplications ranges from 0.05 to 5 mg/kg body weight, preferably 0.1 to2 mg/kg body weight, more preferably 0.1 to 1 mg/kg body weight Atherapeutically effective amount of fusion protein (i.e., an effectivedosage) for the treatment of chronic atheroprogression ranges from 0.5to 6 mg/kg body weight, preferably 1 to 5 mg/kg body weight, morepreferably 2 to 5 mg/kg body weight. The treatment of a subject with atherapeutically effective amount of the fusion protein can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with the fusion protein of theinvention against chronic atheroprogression in the range of between 0.5to 6 mg/kg body weight, preferably 1 to 5 mg/kg body weight, morepreferably 2 to 5 mg/kg body weight, at least twice per week.

Methods to Investigate Platelet-Collagen Interaction and Modulation byInhibitors

Platelet Aggregation and ATP Release

Stimulation of mouse platelet-rich plasma with increasing concentrationsof bovine type I collagen from 0.2 to 4 μg/ml elicits a dose-dependentaggregation from 2 to 95% and a dose-dependent ATP release from 0 to1.66 nM ATP release. A half-maximal collagen concentration was chosenfor further experiments. Incubation of the mouse platelet-rich plasmawith the specific anti-mouse GP VI antibody JAQ 1 (50 μg/ml and 100μg/ml) almost completely abolished platelet aggregation afterstimulation with 2 μg collagen/ml (with 50 μg JAQ 1: 2+/−0.7; with 100μg JAQ 1: 1.5+/−0.3%). Moreover, ATP release was inhibited in anantibody dose-dependent manner to 1.09 nM ATP (10 μg antibody/ml) orcompletely abolished (50 and 100 μg antibody/ml).

Similarly, incubation of mouse platelet-rich plasma with theimmunoadhesin for GP VI (Fc-GPVI-nt) (50 μg/ml and 100 μg/ml) almostcompletely abolished platelet aggregation after stimulation with 2 μgcollagen/ml (with 50 μg Fc-GPVI-nt: 2+/−0.7; with 100 μg Fc-GPVI-nt:1.5+/−0.3%) and ATP release to 0 nM ATP.

Therefore, the immunoadhesin sufficiently inhibited GP VI activation byscavenging the natural GP VI ligand collagen. Both the crucial plateletfunction aggregation and the platelet release mechanism as determined byATP release could be influenced by the Fc-GPVI-nt.

GP VI Mediated Adhesion under Physiological Flow Conditions (FlowChamber)

Adhesion of platelets under physiological shear conditions was tested ina flow chamber. Initial and firm adhesion of platelets was significantlyinhibited by addition of the Fc-GPVI-nt immunoadhesin by 60% (see FIG.4).

GP VI Binding Assay

Adhesion of Fc-GPVI-nt to collagen coated plates was determined in anELISA based fluorescence assay. The binding of the immunoadhesinFc-GPVI-nt dose dependently increased up to saturation levels in aconcentration from 0.2 to 10 μg Fc-GPVI-nt (please see FIG. 5). Thespecificity was demonstrated by comparing binding of Fc-GPVI-nt withthat of the empty immunoadhesin Fc-nt or the uncoated plastic surface(see FIG. 6).

Methods to Investigate Platelet Adhesion and Aggregation at VascularInjury In Vivo as the Crucial Steps for Platelet Activation in AcuteVascular Events

To test the biological significance of platelet-collagen interactions inthe processes of adhesion to lesions in vivo, platelet-vessel wallinteractions following vascular injury of the mouse carotid artery areassessed. Vascular injury to this important vascular bed may serve as amodel for the first steps of arteriosclerosis such as the endotheliallesion in early stage arteriosclerosis or the plaque rupture in laterstages of arteriosclerosis with the unmasking of collagen fibrils fromthe subendothelium. Moreover, this model allows the study of thesubsequent complications of vascular injury. Small endothelial lesionslead to maximal activation of platelets with the following steps ofplatelet adhesion and aggregation. In further steps platelet aggregatescan lead to embolism from the carotid artery with consecutive ischemiccerebral stroke. Thus, this experimental setup serves as a relevant invivo model for a subgroup of patients with unstable atherosclerosisinvolving plaque rupture and endothelial lesions leading to acutecoronary syndrome and stroke.

Vigorous ligation of the carotid artery for 5 min consistently causescomplete loss of the endothelial cell layer and initiates plateletadhesion at the site of injury, as assessed by scanning electronmicroscopy (FIG. 1 a). In vivo fluorescence microscopy may be used todirectly visualize and quantify the dynamic process of plateletaccumulation following vascular injury. Numerous platelets are tetheredto the vascular wall within the first minutes after endothelialdenudation (4620±205 platelets/mm²). Virtually all plateletsestablishing contact with the subendothelium exhibit initially a slowsurface translocation of the “stop-start” type (Savage, B., Saldivar, E.& Ruggeri, Z. M. Initiation of platelet adhesion by arrest ontofibrinogen or translocation on von Willebrand factor. Cell 1996; 84,289-297). While we observed transition from initial slow surfacetranslocation to irreversible platelet adhesion in 88% of all platelets(4182±253 platelets/mm²) (FIG. 1 b), platelet arrest remains transientin only 12% (543±32 platelets/mm²). Once firm arrest is established,adherent platelets recruit additional platelets from the circulation,resulting in aggregate formation (FIG. 1 c). Similar characteristics ofplatelet recruitment are obtained with immobilized collagen in vito. Incontrast, only few platelets are tethered to the intact vascular wallunder physiological conditions (P<0.05 vs. vascular injury) andvirtually 100% of these platelets are displaced from the vascular wallwithout firm arrest (P<0.05 vs. vascular injury, FIG. 1 a-c).

Identification of GP VI as a Novel and Relevant Target Protein inPlatelets for Vascular Injury in Vivo

The high complexity of the platelet-vessel wall interaction whichinvolves a variety of different receptors and signaling pathways makesthe in vivo inhibition of this process very difficult. BesidesGPIb-V-I-X and α_(IIb)β₃ integrin which interact indirectly withcollagen via von Willebrand factor (vWF), a large number of collagenreceptors have been identified on platelets, including most importantlyα₂β₁ integrin (Santoro, S. A. Identification of a 160,000 daltonplatelet membrane protein that mediates the initial divalentcation-dependent adhesion of platelets to collagen. Cell 1986; 46,913-920), GPV (Moog, S. et al. Platelet glycoprotein V binds to collagenand participates in platelet adhesion and aggregation. Blood 2001; 98,1038-1046), and GPVI (Morol, M., Jung, S. M., Okuma, M. & Shinmyozu, K.A patient with platelets deficient in glycoprotein VI that lack bothcollagen-induced aggregation and adhesion. J. Clin. Invest 84,1440-1445). Amidst several reports on different signaling systems whichplay a role in vitro, also GPVI has now been discussed (Gibbins, J. M.,Okuma, M., Farndale, R., Barnes, M. & Watson, S. P. Glycoprotein VI isthe collagen receptor in platelets which underlies tyrosinephosphorylation of the Fc receptor gamma-chain. FEBS Lett. 1997; 413,255-259; Nieswandt, B. et al. Long-term antithrombotic protection by invivo depletion of platelet glycoprotein VI in mice. J. Exp. Med. 2001;193, 459-469, Nieswandt, B. et al. Glycoprotein VI but not α₂β₁ integrinis essential for platelet interaction with collagen. EMBO J. 2001; 20,2120-2130).

To directly test the in vivo relevance of platelet-collagen interactionsin arterial thrombus formation, we inhibited or deleted GPVI in vivo.The monoclonal antibody (mAb) JAQ1 blocks the major collagen-bindingsite on mouse GPVI (Schulte, V. et al. Evidence for two distinctepitopes within collagen for activation of murine platelets. J. Biol.Chem. 2001; 276, 364-368) and almost completely inhibits firm plateletadhesion to immobilized fibrillar collagen under high shear flowconditions (Nieswandt, B. et al. Glycoprotein VI but not alpha2beta1integrin is essential for platelet interaction with collagen. EMBO J.2001; 20, 2120-2130). To study the significance of GPVI-collageninteractions in the dynamic process of platelet adhesion/aggregation inarterial thrombus formation, mice received syngeneic,fluorescence-tagged platelets pre-incubated with JAQ1 Fab fragments orisotype-matched control IgG and carotid injury was induced as describedabove. Very unexpectedly, we found that the inhibition of GPVI reducedinitial platelet tethering following endothelial denudation in thecommon carotid artery by 89% (P<0.05 vs. control IgG, FIG. 2 a), aprocess thought to be mediated mainly by GPIbα interaction withimmobilized vWF (Goto, S., Ikeda, Y., Saldivar, E. & Ruggeri, Z. M.Distinct mechanisms of platelet aggregation as a consequence ofdifferent shearing flow conditions. J. Clin. Invest. 1998; 101, 479-486;Sixma, J. J., van Zanten, G. H., Banga, J. D., Nieuwenhuls, H. K. & deGroot, P. G. Platelet adhesion. Semin. Hematol . 1995; 32, 89-98).Furthermore, stable platelet arrest was reduced by 93% by JAQ1 (FIG. 2a). We observed transition from initial tethering/slow surfacetranslocation to irreversible platelet adhesion in only 58% of thoseplatelets establishing initial contact with the subendothelial surface(compared to 89% with control IgG-pretreated platelets, P<0.05, FIG. 2b). Aggregation of adherent platelets was virtually absent followingpretreatment of platelets with JAQ1 Fab fragments, but not in thecontrols (P<0.05 vs. control, FIGS. 2 c and d). These data demonstratedthat direct platelet-collagen interactions are crucial for initialplatelet tethering and subsequent stable platelet adhesion andaggregation at sites of vascular injury. Furthermore, these findingsshow that GPVI is a key regulator in this process, while other surfacereceptors, most importantly GPIb-V-IX and α₂β₁ are not sufficient toinitiate platelet adhesion and aggregation on the subendotheilum invivo.

To exclude the possibility that this effect is based on stericimpairment of other receptors, e.g. GPIb-V-IX, by surface-bound JAQ1, wegenerated GPVI-deficient mice by injection of JAQ1 five days prior tovascular injury. As reported previously, such treatment inducesvirtually complete loss of GP VI e.g. by internalization and proteolyticdegradation of GPVI in circulating platelets, resulting in a “GPVI knockout”-like phenotype for at least two weeks (Nieswandt, B. et al.Long-term antithrombotic protection by in vivo depletion of plateletglycoprotein VI in mice. J. Exp. Med. 2001; 193, 459-469). Asillustrated in FIG. 3 a, GPVI was undetectable in platelets fromJAQ1-treated mice on day 5 after injection of 100 μg/mouse JAQ1, but notcontrol IgG, while surface expression and function of all other testedreceptors, including GPIb-V-IX, α_(IIb)β₃, and α₂β₁ was unchanged inboth groups of mice, confirming earlier results (data not shown andNieswandt, B. et al. Long-term antithrombotic protection by in vivodepletion of platelet glycoprotein VI in mice. J. Exp. Med. 2001; 193,459-469).

As shown by scanning electron microscopy, platelet adhesion andaggregation following endothelial denudation of the common carotidartery is virtually absent in GPVI-deficient, but not in IgG-pretreatedmice (FIG. 3 b). Next, in vivo video fluorescence microscopy was used todefine platelet adhesion dynamics following vascular injury inGPVI-deficient mice (FIG. 3 c-f) The loss of GPVI significantly reducestethering/slow surface translocation of platelets at the site ofvascular injury (by 83% compared to IgG-pretreated mice. P<0.05). ThisGPVI-independent slow surface translocation requiresvWF-GPIbα-interaction, since it is abrogated by preincubation of theplatelets with Fab fragments of a function blocking mAb against GPIbα(p0p/B) confirming the critical role of GPIbα in this process (notshown). In the absence of GPVI, stable platelet adhesion is reduced byapproximately 90% compared to the (IgG-treated) control, whileaggregation of adherent platelets is virtually absent (FIG. 3 b-f). Wesaw transition from platelet tethering to stable platelet adhesion inonly 58% of all platelets initially tethered to the site of injury(compared to 89% with control mAb-pretreated platelets, P<0.05, FIG. 3d), indicating that GPIbα-dependent surface translocation is notsufficient to promote stable platelet adhesion and subsequentaggregation.

The profound inhibition of platelet tethering by GPVI blockade wassurprising and suggested a previously unrecognized function of thisreceptor in the very initial phase of firm platelet adhesion to vascularlesions. Fibrillar collagen is a major constituent of humanatherosclerotic lesions (Rekhter, M. D. Collagen synthesis inatherosclerosis: too much and not enough. Cardiovasc. Res. 1999; 41,376-384; Rekhter, M. D. et al. Type I collagen gene expression in humanatherosclerosis. Localization to specific plaque regions. Am. J. Pathol,1993; 143, 1634-1648); enhanced collagen synthesis (by intimal smoothmuscle cells and fibroblasts) significantly contributes to luminalnarrowing in the process of atherogenesis (Opsahl, W. P., DeLuca, D. J.& Ehrhart, L. A. Accelerated rates of collagen synthesis inatherosclerotic arteries quantified in vivo. Arterosclerosis 1987; 7,470-476). Plaque rupture or fissuring (either spontaneously or followingballoon angioplasty) results in exposure of collagen fibrils to theflowing blood.

The invention teaches for the first time that such subendothelialcollagens are the major trigger of arterial thrombus formation andreveal an unexpected function of the collagen receptor GPVI in plateletrecruitment to the injured vessel wall. The processes of platelettethering and slow surface translocation under conditions of elevatedshear are known to largely depend on GPIbα interaction with immobilizedvWF. This interaction is, however, not sufficient to establish initialplatelet-vessel wall interactions in vivo as functional GPVI is alsorequired (FIGS. 2 and 3). Thus, both GPIbα and GPVI must act in concertto recruit platelets to the subendothelium. During platelet tethering,ligation of GPVI can shift α_(IIb)β₃ and α₂β₁ integrins from a low to ahigh affinity state. Both α_(IIb)β₃ and α₂β₁ then act in concert topromote subsequent stable arrest of platelets on collagen, whileα_(IIb)β₃ is essential for subsequent aggregation of adherent platelets.Thus, ligation of GPVI during the initial contact between platelets andsubendothelial collagen provides an activation signal that is essentialfor subsequent stable platelet adhesion and aggregation. Importantly,occupation or lateral clustering of GPIbα (during GPIb-dependent surfacetranslocation), which induced low levels of α_(IIb)β₃ integrinactivation in vitro (Kasirer-Friede, A. et al. Lateral clustering ofplatelet GP Ib-IX complexes leads to up-regulation of the adhesivefunction of integrin αIIbβ₃ . J. Biol. Chem. 2002; Vol 277:11949-11956), is not sufficient to promote platelet adhesion in vivo.

The invention therefore has identified an essential receptor forinhibiting platelet attachment to the subendothelium. An antibody whichblocks the interaction of GPVI with exposed collagen can specificallyinhibit all major phases of thrombus formation, i.e. platelet tethering,firm adhesion, and aggregation at sites of arterial injury (e.g. duringacute coronary syndromes). The very profound protection that wasachieved by inhibition or depletion of GPVI establishes the importanceof selective pharmacological modulation of GPVI-collagen interactions tocontrol the onset and progression of pathological atheroscleroticlesions.

Following rupture of the atherosclerotic plaque, exposure ofsubendothelial collagen is the major trigger that initiates plateletadhesion and aggregation at the site of injury, followed by arterialthrombosis (1;24;25). The platelet glycoprotein GPVI, which has beencloned recently (5;6), has been identified by the invention to be themajor platelet collagen receptor (4), mediating platelet adhesion bothin vitro (22) and under (patho-)physiological conditions in vivo (3).Therefore, inhibition of GPVI prevents platelet recruitment and arterialthrombosis in patients with advanced atherosclerosis as shown by thepresent invention by the inhibitory activities of the specific fusionprotein Fc-GPVI-nt on platelet adhesion in Vitro and in vivo.

The Fc-GPVI-nt fusion protein is expressed in HELA cells using anadenoviral expression system to obtain soluble Fc-GPVI-nt.Characterization of the soluble forms of GPVI revealed that Fc-GPVI-ntis secreted as dimer with a molecular mass of approximately 160 kDa.Consistently, Miura and co-workers recently reported that GPVI-Fc-dimeris present as a dimer, in which two GPVI-Fc-dimer molecules arecross-linked by disulfide bonds formed from the Cys in the Fc domain ofeach molecule (21). Importantly, only the dimeric form of GPVI, but notmonomers of the extracellular domain of GPVI, has been reported toexhibit collagen binding affinity and to attenuate collagen-inducedplatelet aggregation (21).

Binding assays were performed to define GPVI-Fc-dimer-collageninteraction. Soluble GPVI binds to immobilized collagen in a saturablemanner. GPVI-Fc-dimer binding to fibrillar collagen was highly specific,since it did not occur to immobilized vWF or BSA. Further, GPVI bindingto immobilized collagen could be inhibited by soluble collagen. Highconcentrations of soluble collagen were required to block GPVI-Fc-dimerbinding, indicating the fusion protein binds immobilized collagen withhigh affinity. Correspondingly, a high association and dissociationconstant (K_(D) approximately 5.8×10⁻⁷ M) has been reported for theGPVI-collagen interaction (21).

Soluble Fc-GPVI-nt has been demonstrated earlier to attenuate plateletactivation and aggregation in response to collagen or convulxin, a snaketoxin, which binds to GPVI with high affinity (6;21;27). Apart fromplatelet aggregation, GPVI is critically involved in the process ofplatelet adhesion to collagen (3;22). In the present study, we,therefore, tested the effects of Fc-GPVI-nt on platelet adhesion underphysiological flow conditions in vitro. We show that soluble Fc-GPVI-ntdose-dependently inhibits platelet adhesion under low and high shearconditions in vitro. In the presence of Fc-GPVI-nt, but not of controlFc peptide, aggregation of adherent platelets was virtually absent,indicating that GPVI contributes to the processes of both plateletadhesion and subsequent activation by immobilized collagen. GPVI conferscollagen responses (i.e. adhesion and aggregation) in a receptordensity-dependent fashion (22). Correspondingly, it has been reportedthat a more than 50% reduction in GPVI expression transfected RBL-2H3cells is associated with a lack of collagen-induced aggregation in thesecells (8;22). Since a low variability in the GPVI receptor density hasbeen reported albeit in a small sample population (22), one might expectthat inhibition of approx. 50% of collagen-GPVI bonds is sufficient toattenuate platelet recruitment to exposed collagen. In the present studydoses of 1 mg/kg Fc-GPVI-nt were required to induce significantinhibition of platelet adhesion underflow, supporting the notion thatmultiple GPVI binding sites are available in each collagen fibril.Similar amounts of a function blocking anti-GPVI antibody were requiredto attenuate platelet-vessel wall injury in vivo (3).

Fibrillar collagen is a major constituent of the normal vessel wall butalso of atherosclerotic lesions (28). Rupture or fissuring of theatherosclerotic plaque results in exposure of collagen fibrils tocirculating platelets. As reported earlier, GPVI-collagen interactionsare essentially involved in arterial thrombus formation followingvascular injury (3). Here we demonstrate the in vivo effects of solubleFc-GPVI-nt on platelet recruitment after arterial injury. Endothelialdenudation was induced by reversible ligation of that carotid artery andthe dynamic process of platelet attachment was monitored by intravitalvideofluorescence microscopy as described (3). We demonstrate for thefirst time in vivo that soluble Fc-GPVI-nt attenuates stable platelettethering, adhesion and platelet aggregation following endothelialdenudation. Inhibition of platelet recruitment by Fc-GPVI-nt wasdose-dependent. Apart from preventing stable arrest of platelets,Fc-GPVI-nt significantly reduced initial platelet tethering/slow surfacetranslocation at sites of endothelial denudation. We have demonstratedearlier that inhibition of GPIbα or of GPVI attenuate platelet tetheringto a similar extent (3), supporting that GPVI and GPIbα interaction needto act in contact to promote platelet tethering to subendothelialcollagen (2;29-31). In fact, the high “on”- and “off”-rates reported forthe GPVI-ligand interaction (22) are consistent with the role of GPVI asa tethering receptor.

The present invention identifies Fc-GPVI-nt as an active ingredient of amedicament to attenuate arterial thrombosis following vascular injury.This concept is further supported by the observation that Fc-GPVI-nt istargeted to the exposed subendothelium at the site of vascular injury,as demonstrated by immunohistochemistry. This implicates that inhibitionof GPVI-collagen interactions are likely to be restricted to the site ofvascular injury, while a prolonged systemic inhibition of plateletfunction is limited by the expected short half-life of unboundFc-GPVI-nt in contrast, administration of monoclonal antibodies directedagainst GPVI inevitably leads to systemic inhibition of GPVI on allcirculating platelets. In addition, Fc-GPVI-nt administration did notaffect platelet counts. In contrast, anti-GPVI mAbs may eventuallyinduce immune thrombocytopenia or a complete loss of GPVI on circulatingplatelets (14;32), hampering their use in clinical practice.Accordingly, Fc-GPVI-nt therapy will likely be associated with a lowerrisk of clinical hemorrhage, compared to anti-GPVI mAb-based strategies.

Platelet adhesion and aggregation at sites of vascular injury is crucialfor hemostasis but may lead to arterial occlusion in the setting ofatherosclerosis and precipitate diseases such as coronary thrombosis andmyocardial infarction. The use of intravenous GPIIb-IIIa receptorinhibitors, has significantly improved the clinical success of patientsundergoing coronary stenting (33-35). However, severe bleedingcomplications have been reported to hamper the outcome of patientstreated with abciximab (36). The present invention demonstrates thatinhibition of GPVI-collagen interactions by Fc-GPVI-nt was sufficient tosignificantly reduce platelet adhesion both in vitro and in vivo;however, the soluble form of GPVI only moderately prolonged tallbleeding times. Similarly, mild bleeding disorders have been reported inpatients with GPVI-deficient platelets (37), indicating that coagulationand hemostasis are effective even in the complete absence of GPVI. Inpart this discrepancy may be due to the fact that inhibition or absenceof GPVI does not interfere with platelet aggregation in response toplatelet agonists other than collagen, e.g. ADP, tissue factor orthrombin. In contrast, direct inhibition of GPIIb-IIIa, e.g. by 7E3 orits humanized derivative, blocks fibrinogen binding to platelets, aprocess which is essential for platelet aggregation, and substantiallyattenuates platelet aggregation to most platelet agonist known thus far.Accordingly, Fc-GPVI-nt therapy are associated with a lower risk ofclinical hemorrhage, compared to anti-GPIIb-IIIa-based strategies.

In conclusion, the present invention provides the first in vivo evidencethat Fc-GPVI-nt attenuates platelet adhesion under flow in vitro andfollowing endothelial denudation in the carotid artery of mice in vivo.This further supports the concept that GPVI-collagen interactions play acentral role in all major phases of thrombus formation, i.e. platelettethering, firm adhesion, and aggregation at sites of arterial injury(e.g. during acute coronary syndromes). The present invention furthersupports the concept that GPVI plays a major role in the progression ofatherosclerosis. Moreover, the present invention shows for the firsttime the causal connection between GPVI and diabetes.

The invention will now be described in further detail with reference tothe following specific examples.

EXAMPLES

Animals. Specific pathogen-free C57BL6/J mice were obtained from CharlesRiver (Suizfeld, Germany). For experiments, 12-weeks-old male mice wereused. All experimental procedures performed on animals were approved bythe German legislation on protection of animals.

Monoclonal antibodies. Monoclonal antibody (mAb) anti GPVI (JAQ 1) andanti GPIbα (p0p/B) and Fab fragments from JAQ and p0p/B were generatedas described (Bergmeier, W., Rackebrandt, K., Schroder, W., Zirngibl, H.& Nieswandt, B. Structural and functional characterization of the mousevon Willebrand factor receptor GPIb-1× with novel monoclonal antibodies.Blood 2000; 95, 886-893; Nieswandt, B., Bergmeier, W., Rackebrandt, K.,Gessner, J. E. & Zirngibl, H. Identfication of critical antigen-specificmechanisms in the development of immune thrombocytopenic purpura inmice. Blood 2000; 96, 2520-2527). Irrelevant control rat IgG wasobtained from Pharmingen (Hamburg, Germany).

Generation of GPVI-Deficient Mice

To generate mice lacking GPVI, C57BL6/J wild-type mice were injectedwith 100 μg JAQ1 i.c. Animals were used for in vivo assessment ofplatelet adhesion on day 5 after mAb injection. Absence of GPVIexpression on platelets was verified by Western blot analysis and flowcytometry.

Flow Cytometry

Heparinized whole blood, obtained from wild type C57BL6/J mice orGPVI-depleted mice was diluted 1:30 with modified Tyrodes-HEPES buffer(134 mM Knack, 0.34 mM Na₂HPO₄, 2.9 mM KCl, 12 mM NaHCO₃, 20 mM HEPES, 5mM glucose, and 1 mM MgCl₂, pH 6.6). The samples were incubated withfluorophore-labeled mAb anti-GPVI (JAQ1) and anti-CD41 for 10 min atroom temperature and directly analyzed on a FACScan™ (Becton Dickinson).

Cloning, viral expression and purification of soluble human and murineGPVI. To generate a soluble form of human GPVI, the extracellular domainof human GPVI was cloned and fused to the human immunoglobin Fc domainaccording to the following examples 1 to 3. Adenoviral constructs codingfor the GPVI-Fc-fusion protein or control Fc were prepared to generatethe recombinant protein. GPVI-Fc and control Fc were expressed assecreted soluble proteins using the human HELA cell line to preventmisfolding and non-glycosylation of the expressed proteins.

Example 1 Cloning of the Immunoadhesin of GP VI (Fc-GPVI-nt)

We generated an immunoadhesin of the GP VI receptor by generating arecombinant fusion protein of the n-terminal part of GP VI—which encodesthe extracellular domain of GPVI—together with the Fc part of an IgG.The Fe was amplified from a human heart cDNA library (Clonetech, PaloAlto, Calif.) by PCR using the forward primer5′-cgcggggcggccgcgagtccaaatcttgtgacaaaac-3′ (SEQ ID No. 4) and thereverse primer 5′-gcgggaagctttcatttacccggagacagggag-3′(SEQ ID No. 5).The PCR reaction was performed at 58° C. annealing temperature and 20cycles with the Expand High Fidelity PCR System (Roche MolecularBiochemicals, Mannheim, Germany). The PCR fragment was cloned in theplasmid pADTrack CMV with NotI/HindIII and the sequence was checked bysequencing (MediGenomix, Martinsried, Germany).

For cloning of the extracellular domain of the human GPVI RNA fromcultured megakaryocytes was isolated (RNeasy Mini Kit; Qiagen, Hilden,Germany) according to the manufacter's protocol and reversetranscription was performed (Omniscript RT Kit; Qiagen) with 2 μg RNA at37° C. overnight. 100 ng of the reaction was used as a template in PCRamplification of the hGPVI with the primer5′-gcggggagatctaccaccatgtctccatccccgacc-3′ (SEQ ID No. 6)and5′-cgcggggcggccgccgttgcccttggtgtagtac-3′(SEQ ID No. 7). The PCR reactionwas performed at 54° C. annealing temperature and 24 cycles with theExpand High Fidelity PCR System (Roche Molecular Biochemicals, Mannheim,Germany). The PCR fragment was cloned in the plasmid pDATrack CMV Fcwith BglII/NotI and the sequence was checked by sequencing.

Construction of a Monomeric Fusion Protein Based on Fc-GPVI-nt

The Fc monomer fragment was amplified by PCR using the primer pair5′-cgcggggcggccgcccagcacctgaactcctg-3′ (SEQ ID No. 8) and5′-cgcggggatatctcatttacccggagacagggag-3′ (SEQ ID No. 9) and pADTrack CMVgpVI-Fc as a template. The PCR reaction was performed at 58° C.annealing temperature and 20 cycles with the Expand High Fidelity PCRSytem (Roche Molecular Biochemicals, Mannheim, Germany). The Fe monomerPCR fragment (NotI/EcoRV) and the gpVI fragment from pADTrack CMVgpVI-Fc (BgIII/NotI) were cloned as described above.

Example 2 Generation of the Adenovirus for Fc-GPVI-nt (Ad-Fc-GPVI-nt)

The plasmid pADTrack CMV Fc-GPVI-nt was linearized with Pmel (NewEngland Biolabs, Beverly, Mass.) overnight, dephosphorylated andpurified (GFX DNA and Gel Purification Kit Amersham Pharmacia Biotech,Uppsala, Sweden). For recombination electrocompetent E. coli BJ5183(Stratagene, La Jolla, Calif.) were cotransformed with 1 μg of thelinearized plasmid and 0.1 μg pAdeasy1 at 2500 V, 200 Ω and 25 μFD (E.coli-pulser, Biorad, Heidelberg, Germany), plated and incubatedovernight at 37° C. The colonies were checked after minipreparation ofthe plasmid-DNA with PacI and the positive clones were retransformed inE. coli DH5α.

For transfection (Effectene Transfection reagent; Qiagen, Hilden,Germany) of 293 cells plasmid-DNA was digested with PacI. The cells werecultured for 7 days and harvested by scraping and centrifugation. Thepellet was resuspended in Dulbecco's PBS and the cells were lysed byfour repetitive freezing (−80° C.) and thawing (37° C.) cycles. Celldebris was removed by centrifugation and the lysate stored at −80° C.

For plaque selection of recombinant virus 293 cells are infected inDulbeccos PBS for 1 hour at room temperature under gentle agitation withdifferent serial dilutions of lysate from transfection. Following theinfection, the cells are overlayed with growth medium containing 0.5%agarose (1:1 mix of modified Eagles medium 2×, Gibco Life Technologies#21935, supplemented with 20% serum, 2× Pencillin/Streptomycin, 2×L-glutamin and agarose in water 1%, Seacam). 5-14 days post infectionthe cell layer was monitored for formation of plaques which were pickedusing a pasteur pipett, resuspended in 0.5 ml Dulbeccos PBS and storedat −80° C. The plaques were used for further amplification rounds on 293cells.

Construction of Human gpVI-Fc Monomer Expressing Stable CHO

The monomer expressing cells were generated in accordance with example2.

Example 3 Fc-GPVI-nt Protein and Fc Control Immunoadhesin Purification

The culture supernatant of Ad-Fc-GPVI-nt-infected Hela cells wascollected 2 days after infection, centrifugated (3800 g, 30 min, 4° C.)and filtrated (0.45 μm). The immunoadhesin was precipitated by additionof 1 vol. ammonium sulfate (761 g/l) and stirred overnight at 4° C. Theproteins were pelleted by centrifugation (3000 g, 30 min, 4° C.),dissolved in 0.1 Vol PBS and dialysed in PBS overnight at 4° C. Theprotein solution was clarified by centrifugation (3000 g, 30 min, 4° C.)and loaded on a protein A column (HiTrap™ protein A HP, AmershamPharmacia Biotech AB, Uppsala, Sweden). The column was washed withbinding buffer (20 mM sodium phoshate buffer pH 7.0, 0.02% NaN₃) untilOD₂₈₀<0.01 and eluted with elution buffer (100 mM glycine pH 2.7). Theeluted fractions were neutralized with neutralisation buffer (1 MTris/HCl pH 9.0, 0.02% NaN₃), pooled, dialysed in PBS overnight at 4°C., aliquotated and frozen at −20° C.

The molecular mass of Fc-GPVI-nt protein was ˜80 kDa under reducingconditions in SDS-PAGE, as detected by Coomassle blue stain or byimmunoblotting with peroxidase-conjugated goat anti-human Fc antibody orby the anti-GPVI mAb 5C4 (FIG. 1 a, upper and middle panel).

In contrast, a ˜160 kDa protein was identified under non-reducingconditions (FIG. 1 a, lower panel), supporting the notion that GPVI-Fcis obtained solely as dimer (21).

Example 4 GP VI Inhibitor Screening Assay

ELISA plates (Immulon2 HB, Dynx Technologies, Chantilly, Va.) werecoated overnight at 4° C. with 1 μg/well collagen (type I bovine; BDBioscience, Bedford, Mass.) in 100 μl 50 mM Tris/HCl pH 8.0. The platewas washed with 250 μl/well PBS/0.05% Tween 20 (PBST) twice and blockedwith 250 μl/well Roti-Block (Roth, Karisruhe, Germany) overnight. Theplate was washed with 250 μl/well PBST twice, 100 μl Fc-GPVI-nt in PBSTwas added (optimal 2 μg/well) and the plate was incubated for 1 h atroom temperature. After 5-fold washing with 250 μl PBST 100 μlperoxidase-conjugated goat anti-human IgG antibody (Dianova, Hamburg,Germany) was added in a dilution of 1:10000 and incubated for 1 h atroom temperature. After repeated washing with 250 μl PBST 100 μldetection reagent (BM Blue POD Substrate; Roche, Mannheim, Germany) wasadded and incubated for 15 min. The reaction was stopped by the additionof 100 μl 1 M H₂SO₄ and the plate was measured at 450 nm against thereference wavelength 690 nm. To screen for potential inhibitors, testcompounds are added to the incubation in 100 μl PBST at variousconcentrations.

Example 5 Platelet Aggregation and Luminometry

Platelet aggregation ex vivo and in vitro was evaluated by opticalaggregometry in citrated blood samples at 37° C. using a two channelChronolog aggregometer (Nobis, Germany). Platelet-rich plasma wasprepared from citrated whole blood by centrifugation (200 g for 20 min).The final platelet count was adjusted to 2×10⁸ platelets/ml withautologous plasma. After adjustment of the baseline, collagen (type 1,bovine) from 0.2 to 4 μg/ml was added and aggregation was recorded for 5min. Simultaneously, release of ATP was recorded using the fireflyluminometer method. Incubation with the monoclonal GP VI antibody JAQ 1was performed for 15 min with 50 μg/ml antibody.

Example 6 In vitro Platelet Adhesion Assay for GP VI/CollagenInteraction

From ACD (20% final concentration) blood platelet rich plasma wasprepared and adjusted to a final concentration of 10⁸ platelets/ml byHepes Tyrode (pH 6.5). Coverslips were coated with monolayers of variousadhesive proteins (Collagen, vWF) at different concentrations. Perfusionstudies were carried out in a perfusion chamber generated from glasscoverslips. Perfusion was performed at shear rates of 500/s representinglow-medium flow and 2000/s representing high shear rates. Adhesion wasmeasured at 37° C. for 20 minutes and then drawn through the chamber atfixed wall shear rates for 5 minutes using an automated syringe pump.After perfusion the coverslips were gently washed with Hepes Tyrode,taken from the chamber. Coverslips were repeatedly washed with HepesTyrode to completely remove adhesive platelets. The platelets insuspension were quantitatively analysed by FACS measurements. Theanalysis of the functional status of platelets was further assessed byanalysis of surface marker expression (CD 41; CD 61 and CD 62 P)according to the standard flow cytometry protocol.

Example 7 Preparation of Platelets for Intravital Microscopy

Platelets (wild type, or GPVI-deficient) were isolated from whole bloodas described (Massberg, S. et al., Platelet-endothelial cellinteractions during ischemia/reperfusion: the role of P-selectin. Blood1998; 92, 507-515) and labeled with 5-carboxyfluorescein diacetatsuccinimidyl ester (DCF). The DCF-labeled platelet suspension wasadjusted to a final concentration of 200×10⁶ platelets/250 μl. Whereindicated, fluorescent wild type platelets were preincubated with 50μg/ml anti-GPVI (JAQ1) Fab fragments, or anti GPIbα (p0p/B) Fabfragments for 10 min. Subsequently, the pretreated platelets togetherwith the Fab fragments were infused into wild type recipient mice andplatelet adhesion was assessed prior to and after carotid injury by invivo video microscopy, as described below.

Example 8 Assessment of Platelet Adhesion and Aggregation by IntravitalMicroscopy

Wild type C57BL6/J or GPVI-deficient mice were anesthetized byintraperitoneal injection of a solution of midazolame (5 mg/kg bodyweight, Ratiopharm, Ulm, Germany), medetomidine (0.5 mg/kg body weight,Pfizer, Karlsruhe, Germany), and fentanyl (0.05 mg/kg body weight,CuraMed Pharma GmbH, Munich, Germany). Polyethylene catheters (Portex,Hythe, England) were implanted into the right jugular vein andfluorescent platelets (200×10⁶/250 μl) were infused intravenously. Theright common carotid artery was dissected free and ligated vigorouslynear the carotid bifurcation for 5 min to induce vascular injury. Priorto and following vascular injury, the fluorescent platelets werevisualized in situ by in vivo video microscopy of the right commoncarotid artery. Platelet-vessel wall interactions were monitored using aZeiss Axiotech microscope (20× water immersion objective, W 20×/0.5,Zeiss) with a 100 W HBO mercury lamp for epi-Illumination. Allvideo-taped images were evaluated using a computer-assisted imageanalysis program (Cap image 7.4, Dr. Zeinti, Heidelberg, Germany).Transiently adherent platelets were defined as cells crossing animaginary perpendicular through the vessel at a velocity significantlylower than the centerline velocity; their numbers are given as cells permm² endothelial surface. The number of adherent platelets was assessedby counting the cells that did not move or detach from the endothelialsurface within 10 seconds. The number of platelet aggregates at the siteof vascular injury was also quantified and is presented per mm².

Example 9 Scanning Electron Microscopy

Following intravital videofluorescence microscopy, the carotid arterywas perfused with PBS (37° C.) for 1 min, followed by perfusion fixationwith phosphate-buffered glutaraldehyde (1% vol/vol). The carotid arterywas excised, opened longitudinally, further fixed by immersion in 1%PBS-buffered glutaraldehyde for 12 hours, dehydrated in ethanol, andprocessed by critical point drying with CO₂. Subsequently, the carotidartery specimens were oriented with the lumen exposed, mounted withcarbon paint sputter coated with platinum, and examined using a fieldemission scanning electron microscope (JSM-6300F, Jeol Ltd., Tokyo,Japan).

Example 10 Assessment of Fc-GPVI-nt Binding to Immobilized Collagen

The binding of Fc-GPVI-nt to immobilized collagen was determined. ELISAplates (Immulon2 HB, Dynx Technologies, Chantilly, Va.) were coated overnight at 4° C. with 1 μg collagen (typI bovine; BD Bioscience, Bedford,Mass.) in 100 μl coating buffer (1.59 gA Na₂CO₃, 2.93 g/l NaHCO₃, 0.2 μlNaN₃, pH 9.6). The plates were washed with 250 μl/well PBS/0.05% Tween20 (PBST) twice and blocked with 250 μl/well Roti-Block (Roth,Karlsruhe, Germany) over night The plates were washed with 250 μl/wellPBST twice, then 3.0, 6.0, 12.5, 25.0, 50.0 or 100 μg/ml Fc-GPVI-nt inPBST was added and the plate was incubated for 1 hr at room temperature.Where indicated, Fc-GPVI-nt (20 μg/ml) was preincubated for 10 min withsoluble collagen. After incubation the plates were washed 5 times with250 μl PBST and peroxidase conjugated goat anti-human IgG antibody Fcγfragment specific (109-035-098; Dianova, Hamburg, Germany) was added ina dilution of 1:10.000 and incubated for 1 hr at room temperature. After5 fold washing with 250 μl PBST 100 μl detection reagent (BM Blue PODSubstrate; Roche, Mannheim, Germany) was added and incubated up to 10min. The reaction was stopped by the addition of 100 μl 1 M H₂SO₄ andthe plate was measured at 450 nm against reference wavelength 690 nm.

Fc-GPVI-nt showed a dose-dependent and saturable binding to immobilizedcollagen (FIG. 9 b). Half maximal collagen binding was observed at afinal Fc-GPVI-nt concentration of 6.0 μg/ml. Binding of GPVI-Fc did notoccur to BSA, vWF (FIG. 9 c, left panel) or Poly-L-Lysin (not shown),supporting the specificity of Fc-GPVI-nt binding. Moreover, we did notdetect any significant binding of the control Fc protein lacking theexternal GPVI domain under identical conditions (FIG. 9 c, right panel).

To further address the specificity of GPVI-binding, we the ability ofsolubilized fibrillar collagen to compete with immobilized collagen forthe association with Fc-GPVI-nt was tested. Soluble collagen inhibitedFc-GPVI-nt-binding to immobilized collagen in a dose-dependent manner(FIG. 9 d). A concentration of 100 μg/ml soluble collagen was requiredto reduce Fc-GPVI-nt binding by more than 50%. Together, these dataindicated that Fc-GPVI-nt binding to collagen is specific andcharacterized by high affinity.

Example 11 Generation of monoclonal antibody against human GPVI.

Monoclonal antibodies were generated essentially as described (17).Lou/C rats were immunized with the adenovirally expressed humanFc-GPVI-nt fusion protein. Screening of hybridoma supernatants wasperformed in a solid-phase immunoassay using Fc-GPVI-nt or FC lackingthe GPVI domain. Screening identified the supernatant of hybridoma 5C4to bind specifically to Fc-GPVI-nt but not to Fc lacking the externalGPVI domain. The immunoglobulin type was determined with rat Ig class(anti-IgM) and IgG subclass-specific mouse mAbs. The monoclonalantibodies were purified using Protein G-Sepharose columns. Antibodyspecificity of 5C4 was verified by immunoblotting against Fc-GPVI-nt andcontrol Fc. 5C4 monoclonal antibody detected adenovirally expressedFc-GPVI-nt but not control Fc. Furthermore, human GPVI was recovered inlysates obtained from human platelets. In addition, 5C4 bindsspecifically to the surface of platelets but not of leukocytes or redblood cells, as demonstrated using flow cytometry (not shown).

Example 12 FACS Measurement of CD62 P Externalisation

Human citrate blood was collected from volunteers. Platelet rich plasma(PRP) was generated after centrifugation and washing procedures (PBS 1×;pH 7.2) with 2000 rpm at 4° C. and resuspension. PRP diluted in stainingbuffer (1×PBS (w/o Ca²⁺ and Mg⁺) with 0,1% sodium azide and 2% fetalbovine serum (FBS), 2 mM CaCl) was incubated with equine collagen type 1(0; 2; 5 and 10 μg/ml; Nobis) in the presence of Fc-GPVI-nt (100 μg/ml)or equimolar concentrations control Fc. Anti CD 62P antibodies labelledwith the fluorophor peroxidase (Immunotech) were added. FACS measurementwas performed with an Becton Dickenson FACScalibur device.

Increasing concentrations of collagen led to platelet secretion fromalpha granules indicated by CD 62P externalisation. Co-incubation ofcollagen with Fc-GPVI-nt blunted the CD62 P externalisation determinedby FACS (FIG. 10).

Example 13 Platelet Aggregation and ATP Release

PRP was generated as described above.

Aggregation was determined in a Whole-Blood-Aggregometer 500VS(Chrono-Log Corporation). Platelet cell number from PRP was adjusted to1.0×10⁸ cells/ml by Thyrodes-HEPES buffer (2.5 mmol/l HEPES, 150 mmol/lNaCl, 12 mmol/l NaHCO₃, 2.5 mmol/l KCl, 1 mmol/l MgCl₂, 2 mmol/l CaCl₂,5.5 mmol D-Glucose, 1 mg/ml BSA, pH 7.4). Chrono-Lume #395 (Chrono-LogCorporation) was added for ATP measurement Agonists were added to theplatelets, pipetted into the aggregometer and aggregation was startedunder defined stirring conditions. Aggregation was determined by changeof light transmission due to coagulating platelets and normalised to aninternal standard. ATP release is determined at the characteristicwavelength of Chrono-Lume for ATP and normalised to an internal standardaccording to the manufacturer's instructions.

Platelet aggregation and ATP release was specifically inhibited byFc-GPVI-nt for collagen mediated agonist stimulation (FIG. 11 a & b).ADP- and thrombin-mediated (TRAP 10 μM) platelet aggregation and ATPrelease was unaffected by Fc-GPVI-nt.

Example 14 PDGF Release from Human Platelets

PRP from human volunteers was prepared as described above. PDGF releasefrom human platelets was determined with a kit system (R & D Systems #DHD00B) according to the manufacturer's instructions. PDGF release wasstimulated with collagen type 1 (20 μg/ml; Nobis) under controlconditions and in the presence of Fc-GPVI-nt (100 μg/ml) or equimolarconcentrations of control Fc. PDGF release is normalised to themanufacturer's standard probe.

PDGF release as an indicator for release of endogenous transmitters fromalpha granules of platelets was also blunted after collagen stimulation.(FIG. 11 c).

Example 15 Effect of Fc-GPVI-nt on Bleeding Time from Human Whole Bloodin vitro

In vitro bleeding time was determined with an PFA-100 device(Dade-Behring). 800 μl of human whole blood was injected in the PFA-100device. Bleeding time was measured with ADP/collagen andepinephrine/collagen coated measuring cells according to themanusfacturer's instructions.

There was no significant prolongation of bleeding time in vito (PFA-100device) with increasing concentrations of Fc-GPVI-nt after differentagonist stimulations. In contrast, therapeutically relevantconcentrations of ReoPro maximally prolonged bleeding time in thePFA-100 device (FIG. 12).

Example 16 Effect of Soluble GPVI on Platelet Adhesion to ImmobilizedCollagen under Flow

Human platelets were isolated from ADC-anticoagulated whole blood asdescribed (18). Washed platelets were resuspended in Tyrodes-HEPESbuffer (2.5 mmol/l HEPES, 150 mmol/l NaCl, 12 mmol/l NaHCO₃, 2.5 mmol/lKCl, 1 mmol/l MgCl₂, 2 mmol/l CaCl₂, 5.5 mmol D-Glucose, 1 mg/ml BSA, pH7.4) to obtain a platelet count of 2×10⁸ cells/ml. Adhesion of plateletsto plates coated with immobilized collagen was determined in a parallelplate flow chamber in the presence of 200 μg/ml Fc-GPVI-nt or controlFc.

GPVI plays a crucial role in the process of platelet recruitment toimmobilized collagen in vitro (22). We determined the effect ofFc-GPVI-nt on adhesion of human platelets to immobilized collagen undershear conditions in vitro. As reported by others earlier (23), plateletsadhered firmly to immobilized collagen at both low (500 sec⁻¹) and high(1000 sec⁻¹) shear rates forming thrombi (FIG. 13). Soluble Fc-GPVI-nt,but not control Fc lacking the external GPVI domain, significantlyattenuated platelet adhesion on immobilized collagen by 37 and 44% atshear rates of 500 sec⁻¹ and 1000 sec⁻¹, respectively (FIG. 13).Inhibition was specific since Fc-GPVI-nt did not affect plateletadhesion to immobilized vWF.

Example 17

Determination of Fc-GPVI-nt plasma concentrations was carried out withan IMMUNO-TEK ELISA system for the quantitative determination of humanIgG (ZeptoMetrix Corporation; Cat # 0801182). Specific peroxidaseconjugated goat anti-human IgG antibodies against the Fc part of theFc-GPVI-nt are used (Dianova). After several washing steps with PBS-Taccording to the manufacturer's specifications peroxidase substrate (BMBlue POD, Roche) is added and measured at the characteristic 450 nmwavelength in an ELISA assay reader (Tecan Sunrise). The Fc-GPVI-ntconcentration is quantified by comparison to an internal human IgGstandard, Fc-GPVI-nt showed favourable in vivo pharmacokinetics. Aftersingle intraperitoneal injection in mice high plasma levels weremeasurable after 24 hours and the half life of the fusion proteinexceeded 96 hours (FIG. 14 a). Repeated intraperitoneal injection wasleading to blood accumulation of the fusion protein (FIG. 14 b)suggesting favourable kinetics for long term application for thetreatment of chronic diseases. After single intravenous injection ofFc-GPVI-nt with increasing doses, dose-dependent plasma concentrationsof Fc-GPVI-nt were detectable over 5 to 60 minutes up to 14 hours (FIG.14 c).

Example 18 Preparation of Murine Platelets for Intravital FluorescenceMicroscopy

Murine platelets were isolated from whole blood and labeled with5-carboxyfluorescein diacetate succinimidyl ester (DCF) as reportedearlier (19). The DCF-labeled platelet suspension was adjusted to afinal concentration of 200×10⁶ platelets/250 μl. Adhesion of murineplatelets was assessed prior to and after carotid injury by in vivovideo microscopy, as described below.

Example 19 Carotid Ligation and Assessment of Platelet Adhesion andAggregation by Intravital Microscopy

Platelet recruitment following endothelial denudation was performed asreported earlier (3). In brief, wild type C57BL6/J mice wereanesthetized by intraperitoneal injection of a solution of midazolame (5mg/kg body weight, Ratiopharm, Ulm, Germany), medetomidine (0.5 mg/kgbody weight, Pfizer, Karisruhe, Germany), and fentanyl (0.05 mg/kg bodyweight, CuraMed Pharma GmbH, Munich, Germany). Where indicated,Fc-GPVI-nt (1 or 2 mg/kg body weight) or control Fc in an amountequimolar to 2 mg/kg Fc-GPVI-nt was administered intravenously.Thereafter, endothelial denudation was induced near the carotidbifurcation by vigorous ligation for 5 min. Following induction ofvascular injury luorescent platelets (200×10⁸/250 μl) were infusedintravenously via polyethylene catheters (Portex, Hythe, England)implanted into the right jugular vein. The fluorescent platelets werevisualized in situ by in vivo video microscopy of the right commoncarotid artery using a Zeiss Axiotech microscope (20× water immersionobjective, W 20×/0.5, Zeiss) with a 100 W HBO mercury lamp forepi-illumination. All video-taped images were evaluated using acomputer-assisted image analysis program (Cap image 7.4, Dr. Zeintl,Heidelberg, Germany (19;20)). Tethered platelets were defined as allcells establishing initial contact with the vessel wall, followed byslow surface translocation (at a velocity significantly lower than thecenterline velocity) or by firm adhesion; their numbers are given ascells per mm² endothellal surface. The number of adherent platelets wasassessed by counting the cells that did not move or detach from theendothelial surface within 10 seconds. The number of platelet aggregatesat the site of vascular injury was also quantified and is presented permm². In addition, the total thrombus area was assessed using Cap image7.4.

Example 20 Scanning Electron Microscopy

Following intravital videofluorescence microscopy, the carotid arterywas perfused with PBS (37° C.) for 1 min in three animals per group,followed by perfusion fixation with phosphate-buffered glutaraldehyde(1% vol/vol). The carotid artery was excised, opened longitudinally,further fixed by immersion in 1% PBS-buffered glutaraldehyde for 12hours, dehydrated in ethanol, and processed by critical point dryingwith CO₂. Subsequently, the carotid artery specimens were oriented withthe lumen exposed, mounted with carbon paint, sputter coated withplatinum, and examined using a field emission scanning electronmicroscope (JSM-6300F, Jeol Ltd., Tokyo, Japan).

Example 21 Assessment of in vivo Fc-GPVI-nt Binding byImmunohistochemistry

Carotid arteries obtained from mice treated with Fc-GPVI-nt were shockfrozen and embedded in cryoblocks (medite, Medizintechnik GmbH,Burgdorf, Germany). The binding of Fc-GPVI-nt to the endothelium andsubendothelium was determined on 5 μm cryostat sections, stained withperoxidase-conjugated goat anti-human IgG antibody Fcγ fragment specific(109-035-098; Dianova, Hamburg, Germany). Carotid arteries obtained fromFc-treated mice served as controls.

Example 22 Effect of Soluble GPVI on Platelet Counts, Bleeding Time andPlatelet Adhesion in vivo

Animals were treated with 2 mg/kg or 4 mg/kg Fc-GPVI-nt or equimolardoses of control Fc lacking the external GPVI domain. Infusion ofFc-GPVI-nt or control Fc even at the highest dose of 4 mg/kg had notsignificant effects on peripheral platelet counts. Moreover, theFc-GPVI-nt fusion protein, did not induce any significant prolongationof tail bleeding times compared to control animals (FIG. 15 a). Theabsolute bleeding times were 1.9±0.9 in PBS treated mice and 2.9±1.9 minand 4.6±0.6 min in mice treated with 2 mg/kg or 4 mg/kg Fc-GPVI-nt. Incontrast, bleeding times were prolonged considerably (42.6±21.6) inIntegrilln-treated animals (0.2 mg per kg IV).

The effects of Fc-GPVI-nt on platelet recruitment in a mouse model ofcarotid injury may be studied using intravital fluorescence microscopy.Animals were treated with 1 mg/kg or 2 mg/kg Fc-GPVI-nt or an equimolaramount of control Fc lacking the external GPVI domain as describedabove. After infusion of Fc-GPVI-nt or control Fc endothelial denudationof the mouse carotid artery was induced by vigorous ligation as reportedpreviously (3). Ligation of the carotid artery consistently causedcomplete loss of the endothelial cell layer. Platelet adhesion wasdirectly visualized and quantified using in vivo fluorescence microscopy(19;20) (FIG. 15 d). In control (Fc-treated) mice numerous plateletswere tethered to the vascular wall within the first minutes afterendothelial denudation (12.026±1.115 tethered platelets/mm²). Plateletsestablishing contact with the subendothelium exhibited initially a slowsurface translocation, which is frequently followed by subsequent firmplatelet adhesion and platelet aggregation (5.494±874 adherentplatelets/mm² and 114±17 platelet thrombil/mm²). In contrast, in thepresence of Fc-GPVI-nt platelet recruitment to the site of vascularinjury was dramatically attenuated. Platelet tethering was reduced by 65and 71% compared to Fc-treated animals following pretreatment with 1mg/kg or 2 mg/kg Fc-GPVI-nt (P<0.05 vs. control). In parallel, firmplatelet adhesion was reduced in a dose-dependent manner (by 49 and 65%following administration of 1 mg/kg or 2 mg/kg Fc-GPVI-nt, respectively;P<0.05 vs. control). Likewise, aggregation of adherent platelets wasvirtually absent in animals treated with 2 mg/kg Fc-GPVI-nt fusionprotein (P<0.05 vs. control Fc, FIG. 15 b-d). Scanning electronmicroscopy also clearly demonstrated that platelet adhesion andaggregation following endothelial denudation of the common carotidartery were virtually absent in Fc-GPVI-nt treated, but not inFC-pretreated mice (FIG. 15 e). To confirm the presence of Fc-GPVI-nt atthe site of injury, the carotid arteries were excised following in vivomicroscopy and processed further for immunohistochemistry usingperoxidase-conjugated goat anti-human IgG antibodies. InFc-GPVI-nt-treated mice Fc-GPVI-nt was detected on at the luminal aspectof the site of vascular damage (FIG. 15 f). Together, these datademonstrate that Fc-GPVI-nt specifically binds to sites of vascularinjury in vivo and prevents subsequent platelet recruitment.

Effect of soluble GPVI on atherosclerosis. 4 weeks old apoE −/− mice(The Jackson Laboratory) consumed a 0.25% cholesterol diet (HarlanResearch diets) for 6 weeks. After 2 weeks 4 apoE −/− mice were injectedwith Fc-GPVI-nt 200 μg per mouse twice weekly with continous cholesteroldiet 4 apoE −/− mice with the similar protocol were injected with thecontrol Fc protein (200 μg) twice weekly and served as control mice. Forassessment of plaque formation the animals were killed and the vasculartree was carefully dissected from the animals. The whole preparations ofthe aortae and carotides were flushed with 0.9% sodium chloride andfixed. The complete vascular preparation was stained with SUDAN III redto assess plaque formation and viewed under a microscope. Treatment ofatherosclerosis prone apoE −/− knockout mice with Fc-GPVI-nt over 4weeks significantly attenuated atheroprogression. (FIG. 16).

Example 23 FACS Measurement of CD61 and CD32 Surface Expression onPlatelets from Diabetic Patients

Human citrate blood was collected from 111 patients suffering fromdiabetes or from 363 non-diabetic patients. Platelet rich plasma (PRP)was generated after centrifugation and washing procedures (PBS lx, pH7.2) with 2000 rpm at 4° C. and resuspension. Anti CD61 and anti CD32antibodies labelled with the fluorophor peroxidase (Immunotech) wereadded or or the anti monoclonal anti-GPVI antibody 4C9 labelled withFITC. FACS measurement was performed with an Becton DickensonFACScalibur device. Surface expression was quantified by fluorescence.Correleation of CD32 fluorescence and 4C9 fluorescence was calculatedwith the correlation coefficient r=0.516.

Statistical Analysis. Comparisons between group means were performedusing Mann-Whitney Rank Sum Test Data represent mean±s.e.m. A value ofP<0.05 was regarded as significant.

Further Disclosure of the Invention.

GPVI is a membrane bound protein expressed at the surface of platelets.Platelets bind to collagen via the extracellular domain of membranebound GPVI The present invention provides a fusion protein. Theinvention includes amongst other things fusion proteins which directlyor indirectly inhibit collagen-induced platelet activation and have alonger plasma half-life than the extracellular domain of GPVI as anisolated protein. Included are fusion proteins containing anantibody-derived sequence, as in the case of proteins containing atleast part of a heavy chain constant region (e.g. at least the hinge)linked to an active sequence (e.g one having the inhibitory functiondescribed in the previous sentence) through a linker. The activesequence may to the N-terminal side of the linker and the antibodyderived sequence to the C-terminal side.

Embodiments of the present invention reside in a protein which binds tocollagen at the site at which the membrane bound GPVI binds to collagen.

Amongst the fusion proteins of the invention are those having an activepart which binds to collagen competitively with platelet-bound GPVIlinked, e.g. at its C-terminus, to a part which prolongs plasmahalf-life, e.g. an antibody derived sequence. Suitably, the two partsare linked through a linker; in embodiments the linker contains ahydrophilic amino acid and in other embodiments it contains a glycine,e.g. a polyglycine sequence. The part which prolongs plasma half lifemay contain at least one cysteine residue, e.g. two cysteine residues,whereby disulphide cross-links may form so that the protein may form adimer.

In one class of proteins the linker comprises the amino acid sequenceGly-X-Z, where Z is a hydrophilic amino acid and X is any amino acid. Insome embodiments, X is selected from Gly and Ala. In some embodiments, Xmay be Gly. In a class of proteins, Z may be selected from Arg, His,Lys, Ser, Thr, Asp, Glu, Tyr, Asn and Gln in some embodiments, Z may beselected from Arg, Ser, Lys and His: particularly Z is Arg.

The 20 common amino acids may be classified as hydrophobic, polar,positively charged and negatively charged as follows.

Hydrophobic Amino Acids

-   -   A=Ala=alanine    -   V=Val=valine    -   I=Ile=isoleucine    -   L=Leu=leucine    -   M=Met=methionine    -   F=Phe=phenylalanine    -   P=Pro=proline    -   W=Trp=tryptophan

Polar (Neutral or Uncharged) Amino Acids

-   -   N=Asn=asparagine    -   C=Cys=cysteine    -   Q=Gln=glutamine    -   G=Gly=glycine    -   S=Ser=serine    -   T=Thr=threonine    -   Y=Tyr=tyrosine

Positively Charged (Basic) Amino Acids

-   -   R=Arg=arginine    -   H=His=histidine    -   K=Lys=lysine

Negatively Charged Amino Acids

-   -   D=Asp=aspartic acid    -   E=Glu=glutamic acid.

Polar and charged amino acids may be considered hydrophilic, e.g.serine, arginine and lysine. One class of disclosed proteins comprises alinker having a hydrophilic amino acid selected from positively chargedamino acids, e.g. arginine or lysine. A further class comprises a linkerhaving a hydrophilic amino acid selected from negatively charged aminoacids. A third class comprises a linker having a hydrophilic amino acidselected from polar uncharged charged amino acids.

The linker may contain additional amino acids to the N-terminal orC-terminal sides of Gly-X-Z, or both sides. Gly is one possible suchamino acid. The linker may include the amino acid sequenceGly-Gly-Arg-Gly (SEQ ID No. 10). Also to be mentioned are linkerscomprising a polyglycine amino acid sequence e.g. containing 2, 3, 4, 5or more contiguous glycine residues. In one embodiment, the linkercomprises the amino acid sequence Gly-Arg-Gly.

In one class of proteins, the linker may comprise the amino acidsequence X₁-Gly-Z, where X₁ is not cysteine. X₁ may be Gly or Ala oranother amino acid with a hydrophobic side chain, e.g. Val, lie, Leu,Met, Phe, Pro or Trp. In other embodiments, X₁ is Asp, Gin, Ser, Thr orTyr The linker may contain additional amino acids to the N-terminal orC-terminal sides of X₁-Gly-Z, or both sides. Gly is one possible suchamino acid.

In one class of proteins, the linker comprises the sequence Gly-Gly-Arg.Gly-Gly-Lys is another linker sequence.

In another class of proteins, the active sequence is at the N-terminalside of the linker and the antibody-derived sequence is at theC-terminal side of the linker. In a sub-class, the linker comprises atleast one hydrophilic amino acid. In embodiments, the hydrophilic aminoacid may be a residue Z as described above. Some linkers are of theamino acid sequence Gly-X-Z as previously described. Included arelinkers containing at least one of the following sequences as well as 0,1, 2, 3, 4, 5 or more additional amino acids:

-   Gly-Gly-Arg;-   Gly-Gly-Ser;-   Gly-Gly-His;-   Gly-Gly-Lys;-   Gly-Lys-Gly;-   Ser-Gly-Arg;-   Arg-Gly-Ser;-   Gly-Arg-Arg;-   Arg-Gly-Gly;-   Gly-Ala-Arg;-   Arg-Gly-Arg; and-   Arg-Gly-Gly-Ser (SEQ ID No. 11).

Included are fusion proteins which comprise an antibody-derivedpolypeptide. In one class of protein, the antibody-derived polypeptideincludes an Ig heavy chain constant part; in a sub-class of protein, theantibody-derived polypeptide includes a hinge region of animmunoglobulin and is functional to prolong the plasma half-life of theprotein beyond that of a fusion protein which does not contain theantibody-derived polypeptide. In another embodiment, theantibody-derived polypeptide includes a hinge region and a CH2 region ofan immunoglobulin. In one embodiment, the antibody-derived polypeptideincludes a hinge region, a CH2 and CH3 region of an immunoglobulin. Theantibody-derived polypeptide may be an Fc domain of an immunoglobulin,for example. Particular proteins comprise an antibody-derivedpolypeptide which is an Fc domain of an IgG molecule.

The IgG may be an IgG1.

In an alternative embodiment, the antibody-derived polypeptide is apolypeptide which has the properties of an Fc domain of an IgG moleculeor a polypeptide which can confer such properties to a fusion protein.Such properties may include, for example, a prolonged serum half-lifeand thus the incorporation of such a sequence into a fusion proteinconfers a prolonged serum half life on the fusion protein as compared toa protein which does not include the antibody-derived polypeptide.

The protein may be a dimer, for example a dimer containing disulfidebonds between Cys residues of two polypeptides. Typically, the dimer isa homodimer but heterodimers are not excluded. The dimer may be of apolypeptide containing at least part of an Fc hinge region, for exampleof a polypetide as described above containing at least a CH2 region anda hinge region, such as CH2, CH3 and hinge regions, for example.

Thus polypeptides comprising conservative substitutions, insertions, ordeletions, but which still retain the biological activity of the fusionprotein of FIG. 7 (or SEQ ID No. 1), are clearly to be understood to bewithin the scope of the invention.

The term “an extracellular domain of GPVI” includes fragments oranalogues thereof which have the biological activity of theextracellular domain of GPVI as herein described. The biologicalactivity of the extracellular domain of GPVI is considered to include,amongst others, collagen binding activity. It will be appreciated thatfragments or analogues of GPVI may have greater or less binding affinityto collagen than the extracellular domain of GPVI, but nonetheless, willin practice have sufficient binding activity for the protein to betherapeutically useful.

The term “an Fc domain of an immunoglobulin” includes fragments oranalogues thereof which have the properties of an Fc domain of animmunoglobulin as herein described.

Polynucleotides of the invention such as those described above,fragments of those polynucleotides, and variants of thosepolynucleotides with sufficient similarity to the non-coding strand ofthose polynucleotides to hybridise thereto e.g. under stringentconditions, are all aspects of the invention. Also included are nucleicacid sequences which differ from those described in the previoussentence by virtue of the degeneracy of the genetic code. Exemplarystringent hybridisation conditions are as follows: hybridisation at 42DEG C in 5×SSC, 20 mM NaPO4, pH 6.8, 50% formamide; and washing at 42DEG C in 0.2×SSC. Those skilled in the art understand that it Isdesirable to vary these conditions empirically based on the length andthe GC nucleotide base content of the sequences to be hybridised, andthat formulae for determining such variation exist. See for exampleSambrook et al, “Molecular Cloning: A Laboratory Manual”, SecondEdition, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1989)A common formula for calculating the stringency conditions required toachieve hybridisation between nucleic acid molecules of a specifiedhomology is:T _(m)=81.5° C.+16.6 Log [Na⁺]+0.41[% G+C]−0.63 (% formamide)

One aspect of the invention pertains to isolated nucleic acid molecules(e.g., cDNAs) comprising a nucleotide sequence encoding a fusion proteincomprising an extracellular domain of GPVI and an Fc domain of animmunoglobulin. In particularly preferred embodiments, the isolatednucleic acid molecule comprises one of the nucleotide sequences setforth in Sequence SEQ ID NO: 2 (FIG. 8) or the coding region or acomplement thereof. In other embodiments, the isolated nucleic acidmolecule comprises a nucleotide sequence which hybridizes to or is atleast about 50%, preferably at least about 60%, more preferably at leastabout 70%, 80% or 90%, and even more preferably at least about 95%, 96%,97%, 98%, 99% or more homologous to a nucleotide sequence as in SEQ IDNO: 2 (FIG. 8), or a portion thereof. In other preferred embodiments,the isolated nucleic acid molecule encodes one of the amino acidsequences set forth in SEQ ID NO: 1 (FIG. 7).

In an embodiment, the fusion protein is expressed in CHO (Chinesehamster ovary) cells.

The disclosure further includes the subject matter of the followingparagraphs:

-   1. A fusion protein comprising:    -   a) a first polypeptide which is capable of inhibiting adhesion        of platelets to collagen;    -   b) a second, antibody-derived polypeptide; and    -   c) a linker comprising an amino acid sequence Gly-X-Z or Z-P-Q,        wherein X is an amino acid, P and Q are each independently amino        acids provided that at least one of P and Q is Gly, and Z is a        hydrophilic amino acid.-   2. The fusion protein of Paragraph 1, wherein the first polypeptide    is capable of binding to collagen.-   3. The fusion protein of Paragraph 1 or Paragraph 2, wherein the    first polypeptide comprises an extracellular domain of GPVI or a    variant thereof that is functional for binding to collagen.-   4. The fusion protein of any preceding Paragraph, wherein the first    polypeptide binds to collagen competitively with platelet-bound    GPVI.-   5. The fusion protein of any preceding Paragraph, wherein the first    polypeptide is functional for binding to collagen at the    platelet-bound GPVI binding site of collagen-   6. The fusion protein of any preceding Paragraph, wherein the second    polypeptide comprises an amino acid sequence of an Ig heavy chain    constant part.-   7. The fusion protein of any preceding Paragraph, wherein the second    polypeptide comprises a hinge region of an immunoglobulin and is    functional to prolong the plasma half-life beyond that of a protein    consisting of the first polypeptide and the linker.-   8. The fusion protein of any preceding Paragraph, wherein the second    polypeptide comprises a hinge region and a CH2 region of an    immunoglobulin.-   9 The fusion protein of any preceding Paragraph, wherein the second    polypeptide comprises a hinge region, a CH2 region and a CH3 region    of an immunoglobulin.-   10. The fusion protein of any preceding Paragraph, wherein the first    polypeptide comprises an extracellular domain of GPVI or a variant    thereof that is functional for binding to collagen; and the second    polypeptide comprises an Fc domain of an immunoglobulin or a    functional conservative part thereof.-   11. The fusion protein of any preceding Paragraph wherein Z is    selected from the group consisting of Arg, Ser, Thr, Asp, Glu, Tyr,    Asn and Gin.-   12. The fusion protein of any preceding Paragraph, wherein Z is Ser.-   13. The fusion protein of any preceding Paragraph, wherein Z is Arg.-   14. The fusion protein of any preceding Paragraph, wherein the    linker comprises the amino acid sequence Gly-X-Z and X is Gly.-   15. The fusion protein of any preceding Paragraph, wherein P is Gly.-   16. The fusion protein of any preceding Paragraph, wherein the    linker comprises a diglycine sequence.-   17. The fusion protein of any preceding Paragraph, which is    expressed in a mammalian cell.-   18. A fusion protein comprising:    -   a) an amino acid sequence which, when the protein is        administered, results in inhibition of adhesion of platelets to        collagen; and    -   b) an antibody-derived amino acid sequence, wherein sequence (a)        is linked at its C-terminus to the N-terminus of sequence (b)        through a linker comprising a hydrophilic amino acid.-   19. The fusion protein of Paragraph 18, wherein the antibody-derived    amino acid sequence is an Fc domain of an immunoglobulin.-   20. The fusion protein of Paragraph 18 or Paragraph 19, wherein the    amino acid sequence which results in the inhibition of adhesion of    platelets to collagen is an extracellular domain of GPVI.-   21. The fusion protein of any of Paragraphs 18 to 20, wherein the    hydrophilic amino acid is selected from Arg, Ser, Thr, Lys, His,    Glu, Asp and Asn.-   22. The fusion protein of any of Paragraphs 18 to 21, wherein the    hydrophilic amino acid is Arg.-   23. A fusion protein comprising:    -   a) an amino acid sequence comprising an extracellular domain of        GPVI or a variant thereof that is functional for binding to        collagen;    -   b) a linker comprising an amino acid sequence Gly-Gly-Z, wherein        Z is a hydrophilic amino acid; and    -   c) an amino acid sequence comprising an Fc domain of an        immunoglobulin or a functional conservative part thereof.-   24. The fusion protein of Paragraph 23, wherein the amino acid    sequence (a) is encoded by:    -   (i) a nucleic acid sequence of bases 1 to 807 of SEQ ID No. 2        (FIG. 8);    -   (ii) a nucleic acid sequence which hybridises to bases 1 to 807        of SEQ ID No. 2 (FIG. 8); or    -   (iii) a nucleic acid sequence which differs from bases 1 to 807        of SEQ ID No. 2 (FIG. 8) by virtue of the degeneracy of the        genetic code.-   25. The fusion protein of Paragraph 23 or Paragraph 24, wherein    amino acid sequence (b) is encoded by:    -   (i) a nucleic acid sequence of bases 808 to 816 of SEQ ID. No 2        (FIG. 8);    -   (ii) a nucleic acid sequence which hybridises to bases 808 to        816 of SEQ ID No. 2 (FIG. 8);    -   (iii) a nucleic acid sequence which differs from bases 808 to        816 of SEQ ID No. 2 (FIG. 8) by virtue of the degeneracy of the        genetic code-   26. The fusion protein of any of Paragraphs 23 to 25, wherein the    hydrophilic amino acid is selected from Arg, His and Lys.-   27. The fusion protein of any of Paragraphs 23 to 26, wherein amino    acid sequence (c) is encoded by:    -   (i) a nucleic acid sequence of bases 817 to 1515 of SEQ ID. No.        2 (FIG. 8);    -   (ii) a nucleic acid sequence which hybridises to bases 817 to        1515 of SEQ ID No. 2 (FIG. 8); or    -   (iii) a nucleic acid sequence which differs from bases 817 to        1515 of SEQ ID No. 2 (FIG. 8) by virtue of the degeneracy of the        genetic code.-   28. A protein having the characteristics of a protein obtained by    expressing in a mammalian cell under non-reducing conditions a DNA    sequence comprising in a 5′ to 3′ direction:    -   (i) a DNA sequence comprising bases 1 to 807 of SEQ ID No. 2        (FIG. 8) or a variant thereof in which said sequence encoded by        bases 1 to 807 of SEQ ID No. 2 (FIG. 8) is replaced by a variant        having collagen binding activity,    -   (ii) a DNA sequence comprising bases 808 to 816 of SEQ ID. No. 2        (FIG. 8); and    -   (iii) a DNA sequence comprising bases 817 to 1515 of SEQ ID No.        2 (FIG. 8) or a variant thereof in which said sequence encoded        by bases 817 to 1515 of SEQ ID No. 2 (FIG. 8) is replaced by a        variant having the properties of an Fc region.-   29. A dimer of a polypeptide, the polypeptide comprising    -   a) an extracellular domain of GPVI or a variant thereof that is        functional for binding to collagen; and    -   b) a linker comprising an amino acid sequence Gly-Gly-Z, wherein        Z is a hydrophilic amino acid; and    -   c) an Fc domain of an immunoglobulin or a functional        conservative part thereof.-   30. A method of treating or preventing thrombosis in a subject,    comprising administering to the subject a therapeutically effective    amount of a fusion protein comprising:    -   a) an amino acid sequence which, when the protein is        administered, results in Inhibition of collagen-induced platelet        activation; and    -   b) a antibody-derived amino acid sequence, wherein sequence (a)        is linked at its C-terminus to the N-terminus of sequence (b)        through a linker comprising a hydrophilic amino acid.-   31. The method of Paragraph 30, wherein amino acid sequence (a)    comprises an extracellular domain of GPVI or a variant thereof that    is functional for binding to collagen; and the antibody-derived    amino acid sequence comprises an Fc domain of an immunoglobulin or a    functional conservative part thereof.-   32. The method of Paragraph 30 or Paragraph 31, wherein the fusion    protein comprises sequentially in an N-terminus to C-terminus    direction, a first amino acid sequence, a second amino acid sequence    and a third amino acid sequence wherein said first amino acid    sequence comprises:    -   A) i) an amino acid sequence encoded by a nucleic acid sequence        of bases 1 to 807 of SEQ ID No. 2 (FIG. 8);        -   ii) an amino acid sequence encoded by a nucleic acid            sequence which hybridises to bases 1 to 807 of SEQ ID No. 2            (FIG. 8) and which codes for a polypeptide which binds to            collagen; or        -   iii) an amino acid sequence encoded by a nucleic acid            sequence which differs from bases 1 to 807 of SEQ ID No 2            (FIG. 8) by virtue of the degeneracy of the genetic code and            which binds to collagen;            and wherein the second amino acid comprises    -   B) i) an amino acid sequence encoded by a nucleic acid sequence        of bases 808 to 816 of SEQ ID No. 2 (FIG. 8);        -   ii) a 3-mer amino acid sequence containing a hydrophilic            amino acid or        -   iii) an amino acid sequence encoded by a nucleic acid            sequence which differs from bases 808 to 816 of SEQ ID No. 2            (FIG. 8) by virtue of the degeneracy of the genetic code;            wherein the third amino acid sequence comprises:    -   C) i) an amino acid sequence encoded by a nucleic acid sequence        of bases 817 to 1515 of SEQ ID. No. 2 (FIG. 8);        -   ii) an amino acid sequence encoded by a nucleic acid            sequence which hybridises to bases 817 to 1515 of SEQ ID No.            2 (FIG. 8) and which codes for a polypeptide which is            functional as an Fc domain of an immunoglobulin; or        -   iii) an amino acid sequence encoded by a nucleic acid            sequence which differs from bases 817 to 1515 of SEQ ID No.            2 (FIG. 8) by virtue of the degeneracy of the genetic code            and which is functional as an Fc domain of an            immunoglobulin.-   33 A dimer of a polypeptide, the polypeptide comprising:    -   a) a first polypeptide which is capable of inhibiting adhesion        of platelets to collagen;    -   b) a second, antibody-derived polypeptide; and    -   c) a linker comprising an amino acid sequence Gly-X-Z or Z-P-Q,        wherein X is an amino acid, P and Q are each independently amino        acids provided that at least one of P and Q is Gly, and Z is a        hydrophilic amino acid.-   34. A dimer of a polypeptide, the polypeptide comprising:    -   a) a first polypeptide which is capable of inhibiting adhesion        of platelets to collagen;    -   b) a second, antibody-derived polypeptide; and    -   c) a linker comprising an amino acid sequence Gly-X-Z or Z-P-Q,        wherein X is an amino acid, P and Q are each independently amino        acids provided that at least one of P and Q is Gly, and Z is a        hydrophilic amino acid,        wherein the first polypeptide binds to collagen competitively        with platelet-bound GPVI, the second polypeptide comprises a        hinge region of an immunoglobulin and is functional to prolong        the plasma half-life beyond that of a protein consisting of the        first polypeptide and the linker and wherein the linker        comprises the amino acid sequence Gly-X-Z, wherein Z is selected        from the group consisting of Arg, Ser, Thr, Asp, Glu, Tyr, Asn        and Gin.-   35. A dimer of a polypeptide, the polypeptide comprising:    -   a) a first polypeptide which is capable of inhibiting adhesion        of platelets to collagen;    -   b) a second, antibody-derived polypeptide; and    -   c) a linker comprising an amino acid sequence Gly-X-Z, wherein X        is Gly and Z is selected from the group consisting of Lys and        Arg,        wherein the first polypeptide binds to collagen competitively        with platelet-bound GPVI and wherein the second polypeptide        comprises a hinge region of an immunoglobulin and is functional        to prolong the plasma half-life beyond that of a protein        consisting of the first polypeptide and the linker.

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1. A protein obtained by expressing a DNA sequence in a mammalian cellwherein the DNA sequence comprises, in a 5′ to 3′ direction: (i) a DNAsequence comprising bases 1 to 807 of SEQ ID NO: 2 having collagenbinding activity; (ii) a DNA sequence comprising bases 808 to 816 of SEQID NO: 2; and (iii) a DNA sequence comprising bases 817 to 1515 of SEQID NO: 2, wherein said DNA sequence encodes an amino acid sequence whichcomprises a hinge region of an immunoglobulin, the hinge regioncontaining at least two cysteines, and is functional to prolong plasmahalf life of the protein beyond that of a protein whose amino acidsequence consists of an amino acid sequence encoded by said DNAsequences (i) and (ii); wherein said protein binds collagen.
 2. Adimeric protein of two polypeptide monomers, wherein the amino acidsequence of each polypeptide monomer is the sequence of SEQ ID NO:
 1. 3.The dimeric protein of claim 2, wherein said protein is glycosylated. 4.A fusion protein whose amino acid sequence comprises the sequence of SEQID NO:
 1. 5. A fusion protein whose amino acid sequence consists of thesequence of SEQ ID NO:
 1. 6. A composition comprising the dimericprotein of claim 2 and a pharmaceutically acceptable carrier.
 7. Aprotein obtained by expressing a DNA sequence consisting of the sequenceof SEQ ID NO: 2 in a CHO cell.